Anti-cd137 antibodies and methods of use thereof

ABSTRACT

The instant disclosure provides antibodies that specifically bind to CD137 (e.g., human CD137) and increases CD137 function. Also provided are pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating a subject using these antibodies.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/951,950, filed Apr. 12, 2018, which claims the benefit of U.S.Provisional Application No. 62/485,365, filed Apr. 13, 2017, which isincorporated by reference herein in its entirety.

SEQUENCE LISTING

The sequence listing attached herewith named“404301_AGBW_100USD2_188317_Sequence_Listing.txt” (163,801 bytes) andcreated on Apr. 12, 2018, is herein incorporated by reference in itsentirety.

1. FIELD

The instant disclosure relates to antibodies that specifically bind toCD137 (e.g., human CD137) and methods of using the same.

2. BACKGROUND

CD137, also known as TNFRSF9 or 4-1BB, is a transmembrane protein in theTumor Necrosis Factor (TNF) receptor superfamily. It has an N-terminalextracellular domain containing cysteine-rich motifs, a transmembranedomain, and a short C-terminal cytoplasmic domain containing potentialphosphorylation sites. CD137 is expressed on activated CD4⁺ Tlymphocytes, activated CD8⁺ T lymphocytes, activated natural killer (NK)cells, monocytes, dendritic cells, B cells, neutrophils, and mast cells(Vinay et al. (2011) Cellular & Molecular Immunology 8:281-84). CD137L,also known as TNFSF9 or 4-1BBL, is a ligand of CD137. Upon CD137Lbinding, CD137 transduces a co-stimulatory signal that promotes cellsurvival, proliferation, cytokine production, and activation of effectorfunctions. CD137L binding to CD137 has also been shown to co-stimulateCD8+ T cells to a greater degree than CD4+ T cells.

Studies in animal models have shown that ligation of CD137, using eitherCD137L or agonistic antibodies, suppresses tumor growth by promoting Tcell activity (Vinay et al. (2012) Mol. Cancer. Ther. 11:1062-70). CD137has also been shown to enhance T cell immunity against humanimmunodeficiency virus (HIV) and hepatitis C virus (HCV) followingvaccination (Munks et al. (2004) Immunology 112:559-66; Arribillaga etal. (2005) Vaccine 23:3493-99). Additionally, CD137 agonists have beenshown to ameliorate autoimmunity in animal models of lupus,collagen-induced arthritis, and experimental autoimmuneencephalomyelitis.

Given the apparent role of human CD137 in modulating immune responses,therapeutic agents designed to promote CD137 signaling hold greatpromise for the treatment of diseases that involve immune suppression.

3. SUMMARY

The instant disclosure provides antibodies that specifically bind toCD137 (e.g., human CD137 or cynomolgus CD137) and increase or promoteCD137 function, e.g., CD137-mediated immune activation. Also providedare pharmaceutical compositions comprising these antibodies, nucleicacids encoding these antibodies, expression vectors and host cells formaking these antibodies, and methods of treating a subject using theseantibodies. The antibodies disclosed herein are particularly useful forincreasing T cell activation against an antigen (e.g., a tumor antigenor an infectious disease antigen) and/or decreasing Treg-mediated immunesuppression, and hence for treating cancer in a subject or treating orpreventing an infectious disease in a subject.

Accordingly, in one aspect, the instant disclosure provides an antibodyor isolated antibody comprising a heavy chain variable region (VH)comprising complementarity determining regions (CDRs) CDRH1, CDRH2 andCDRH3 and a light chain variable region (VL) comprising complementaritydetermining regions CDRL1, CDRL2 and CDRL3, wherein:

(a) CDRH1 comprises the amino acid sequence of (SEQ ID NO: 82)X₁X₂X₃X₄H,

wherein

X₁ is G, A, D, E, L, N, Q, R, S, or W;

X₂ is Y, F, H, N, R, or S;

X₃ is Y or H; and

X₄ is M, I, T, or V;

(b) CDRH2 comprises the amino acid sequence of (SEQ ID NO: 2)WINPNSGGTNYAQKFQG; (c) CDRH3 comprises the amino acid sequence of(SEQ ID NO: 83) X₁PX₂YX₃GX₄GLX₅X₆,

wherein

X₁ is E or G;

X₂ is G, A, R, or S;

X₃ is Y, F, H, or S;

X₄ is S, A, or T;

X₅ is D or G; and

X₆ is Y or H;

(d) CDRL1 comprises the amino acid sequence of (SEQ ID NO: 4)GGDDIGDKRVH; (e) CDRL2 comprises the amino acid sequence of(SEQ ID NO: 5) EDRYRPS; and/or(f) CDRL3 comprises the amino acid sequence of (SEQ ID NO: 84)QX₁WX₂X₃X₄X₅X₆X₇PGV,

wherein

X₁ is V or I;

X₂ is D, A, E, G, H, N, or Y;

X₃ is S, A, E, F, L, P, R, T, W, or Y;

X₄ is S, A, L, M, or R;

X₅ is S, A, F, G, L, P, Q, R, or T;

X₆ is D, E, H, V, or Y; and

X₇ is H or Y.

In certain embodiments,

(a) CDRH1 comprises the amino acid sequence of (SEQ ID NO: 85) X₁X₂YX₃H,wherein

X₁ is G, A, D, L, R, S, or W;

X₂ is Y, F, H, or N; and

X₃ is M or V;

(b) CDRH3 comprises the amino acid sequence of (SEQ ID NO: 86)EPGYX₁GX₂GLDX₃,wherein

X₁ is Y or F;

X₂ is S or T; and

X₃ is Y or H; and/or

(c) CDRL3 comprises the amino acid sequence of (SEQ ID NO: 87)QVWX₁X₂X₃X₄X₅X₆PGV,wherein

X₁ is D, A, E, H, N, or Y;

X₂ is S, A, E, L, R, or T;

X₃ is S, A, L, or R;

X₄ is S, A, F, G, L, P, Q, or R;

X₅ is D, E, or V; and

X₆ is H or Y.

In certain embodiments,

(a) CDRH1 comprises the amino acid sequence of (SEQ ID NO: 1) GYYMH;(b) CDRH3 comprises the amino acid sequence of (SEQ ID NO: 3)EPGYYGSGLDY or (SEQ ID NO: 59) EPGYYGTGLDY;  and/or(c) CDRL3 comprises the amino acid sequence of (SEQ ID NO: 6)QVWDSSSDHPGV, (SEQ ID NO: 60) QVWNSSSDHPGV, (SEQ ID NO: 61)QVWDSSSDYPGV, or (SEQ ID NO: 62) QVWYSSPDHPGV.

In certain embodiments, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3comprise the amino acid sequences set forth in SEQ ID NOs: 1, 2, 3, 4,5, and 6; 1, 2, 59, 4, 5, and 6; 1, 2, 3, 4, 5, and 60; 1, 2, 3, 4, 5,and 61; or 1, 2, 3, 4, 5, and 62, respectively.

In certain embodiments, the antibody comprises a VH comprising an aminoacid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to the amino acid sequence of SEQ ID NO: 7. In certainembodiments, the VH comprises the amino acid sequence of SEQ ID NO: 7,63, 64, or 65. In certain embodiments, the VH comprises the amino acidsequence of SEQ ID NO: 7. In certain embodiments, the amino acidsequence of the VH consists of the amino acid sequence of SEQ ID NO: 7,63, 64, or 65. In certain embodiments, the amino acid sequence of the VHconsists of the amino acid sequence of SEQ ID NO: 7. In certainembodiments, X is glutamine (Q). In certain embodiments, X ispyroglutamate (pE). In certain embodiments, the antibody comprises a VLcomprising an amino acid sequence that is at least 75%, 80%, 85%, 90%,95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.In certain embodiments, the VL comprises the amino acid sequence of SEQID NO: 8, 66, 67, or 68. In certain embodiments, the VL comprises theamino acid sequence of SEQ ID NO: 8. In certain embodiments, the aminoacid sequence of the VL consists of the amino acid sequence of SEQ IDNO: 8, 66, 67, or 68. In certain embodiments, the amino acid sequence ofthe VL consists of the amino acid sequence of SEQ ID NO: 8.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to CD137 (e.g., human CD137 or cynomolgusCD137), the antibody comprising a VH comprising the amino acid sequenceof SEQ ID NO: 7, 63, 64, or 65. In certain embodiments, the VH comprisesthe amino acid sequence of SEQ ID NO: 7. In certain embodiments, theamino acid sequence of the VH consists of the amino acid sequence of SEQID NO: 7, 63, 64, or 65. In certain embodiments, the amino acid sequenceof the VH consists of the amino acid sequence of SEQ ID NO: 7. Incertain embodiments, X is glutamine (Q). In certain embodiments, X ispyroglutamate (pE).

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to CD137 (e.g., human CD137 or cynomolgusCD137), the antibody comprising a VL comprising the amino acid sequenceof SEQ ID NO: 8, 66, 67, or 68. In certain embodiments, the VL comprisesthe amino acid sequence of SEQ ID NO: 8. In certain embodiments, theamino acid sequence of the VL consists of the amino acid sequence of SEQID NO: 8, 66, 67, or 68. In certain embodiments, the amino acid sequenceof the VL consists of the amino acid sequence of SEQ ID NO: 8.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to CD137 (e.g., human CD137 or cynomolgusCD137), the antibody comprising a VH and a VL comprising the amino acidsequences of SEQ ID NO: 7 and 8; 63 and 8; 64 and 66; 7 and 67; or 65and 68, respectively. In certain embodiments, the amino acid sequencesof the VH and VL consist of the amino acid sequences of SEQ ID NO: 7 and8; 63 and 8; 64 and 66; 7 and 67; or 65 and 68, respectively. In certainembodiments, X in SEQ ID NO: 7, 63, 64, or 65 is glutamine (Q). Incertain embodiments, X in SEQ ID NO: 7, 63, 64, or 65 is pyroglutamate(pE).

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to CD137 (e.g., human CD137 or cynomolgusCD137), the antibody comprising a VH having an amino acid sequencederived from a human IGHV1-2*02 germline sequence. In certainembodiments, the VH comprises the amino acid sequence set forth in SEQID NO: 3 or 59.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to CD137 (e.g., human CD137 or cynomolgusCD137), the antibody comprising a VL having an amino acid sequencederived from a human IGLV3-21*02 germline sequence. In certainembodiments, the VL comprises the amino acid sequence set forth in SEQID NO: 6, 60, 61, or 62.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to CD137 (e.g., human CD137 or cynomolgusCD137), the antibody comprising a VH having an amino acid sequencederived from a human IGHV1-2*02 germline sequence, and a VL having anamino acid sequence derived from a human IGLV3-21*02 germline sequence.In certain embodiments, the VH comprises the amino acid sequence setforth in SEQ ID NO: 3, and the VL comprises the amino acid sequence setforth in SEQ ID NO: 6, 60, 61, or 62.

In certain embodiments, the antibody comprises a heavy chain constantregion selected from the group consisting of human IgG1, IgG2, IgG3,IgG4, IgA1, and IgA2.

In certain embodiments, the antibody comprises an IgG1 heavy chainconstant region. In certain embodiments, the antibody comprises a heavychain constant region comprising the amino acid sequence of SEQ ID NO:15. In certain embodiments, the amino acid sequence of the IgG1 heavychain constant region comprises an N297A mutation, numbered according tothe EU numbering system. In certain embodiments, the antibody comprisesa heavy chain constant region comprising the amino acid sequence of SEQID NO: 16. In certain embodiments, the amino acid sequence of the IgG1heavy chain constant region comprises S267E and L328F mutations,numbered according to the EU numbering system. In certain embodiments,the antibody comprises a heavy chain constant region comprising theamino acid sequence of SEQ ID NO: 17.

In certain embodiments, the antibody comprises an IgG2 heavy chainconstant region. In certain embodiments, the antibody comprises a heavychain constant region comprising the amino acid sequence of SEQ ID NO:18. In certain embodiments, the amino acid sequence of the IgG2 heavychain constant region comprises an N297A mutation, numbered according tothe EU numbering system. In certain embodiments, the antibody comprisesa heavy chain constant region comprising the amino acid sequence of SEQID NO: 19.

In certain embodiments, the antibody comprises an IgG4 heavy chainconstant region. In certain embodiments, the amino acid sequence of theIgG4 heavy chain constant region comprises an S228P mutation, numberedaccording to the EU numbering system. In certain embodiments, theantibody comprises a heavy chain constant region comprising the aminoacid sequence of SEQ ID NO: 20.

In certain embodiments, the antibody comprises a heavy chain constantregion that is a variant of a wild type heavy chain constant region,wherein the variant heavy chain constant region binds to an FcγR withhigher affinity than the wild type heavy chain constant region binds tothe FcγR. In certain embodiments, the FcγR is FcγRIIB

In certain embodiments, the antibody comprises a light chain constantregion comprising the amino acid sequence of SEQ ID NO: 22.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to CD137 (e.g., human CD137 or cynomolgusCD137), the antibody comprising: (a) a heavy chain comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 9-14,49-54, and 73-78; and/or (b) a light chain comprising the amino acidsequence of SEQ ID NO: 21 and 79-81. In certain embodiments, the heavychain and light chain comprise the amino acid sequences of SEQ ID NOs: 9and 21; 10 and 21; 11 and 21; 12 and 21; 13 and 21; 14 and 21; 49 and21; 50 and 21; 51 and 21; 52 and 21; 53 and 21; 54 and 21; 73 and 21; 74and 21; 75 and 79; 76 and 79; 9 and 80; 49 and 80; 77 and 81; or 78 and81, respectively. In certain embodiments, the heavy chain and lightchain comprise the amino acid sequences of SEQ ID NOs: 9 and 21; or 49and 21, respectively. In certain embodiments, the amino acid sequencesof the heavy chain and light chain consist of the amino acid sequencesof SEQ ID NOs: 9 and 21; 10 and 21; 11 and 21; 12 and 21; 13 and 21; 14and 21; 49 and 21; 50 and 21; 51 and 21; 52 and 21; 53 and 21; 54 and21; 73 and 21; 74 and 21; 75 and 79; 76 and 79; 9 and 80; 49 and 80; 77and 81; or 78 and 81, respectively. In certain embodiments, the aminoacid sequences of the heavy chain and light chain consist of the aminoacid sequences of SEQ ID NOs: 9 and 21; or 49 and 21, respectively. Incertain embodiments, X is glutamine (Q). In certain embodiments, X ispyroglutamate (pE).

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to CD137 (e.g., human CD137 or cynomolgusCD137), wherein binding of the antibody to the CD137 increases the levelof dimerization between the CD137 and a second human CD137 moleculerelative to the level of the dimerization in the absence of theantibody. In certain embodiments, the binding of the antibody to theCD137 increases the level of pairwise binding between the PLAD domainsof the two CD137 molecules relative to the level of pairwise bindingbetween the PLAD domains of the two CD137 molecules in the absence ofthe antibody. In certain embodiments, the binding of the antibody to theCD137 increases the level of pairwise binding between a first region ofthe CD137 molecule and a second region of the second human CD137molecule relative to the level of pairwise binding between the firstregion and the second region in the absence of the antibody, wherein thefirst region and/or the second region comprises the amino acid sequenceof SEQ ID NO: 34.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to CD137 (e.g., human CD137 or cynomolgusCD137), wherein binding of the antibody to CD137 increases the level ofCD137 multimerization (e.g., dimerization) relative to the level ofCD137 multimerization (e.g., dimerization) in the absence of theantibody. In certain embodiments, the increase in the level of CD137multimerization (e.g., dimerization) comprises an increase in the levelof pairwise binding between the PLAD domains of two CD137 molecules. Incertain embodiments, the increase in the level of CD137 multimerization(e.g., dimerization) comprises an increase in the level of pairwisebinding between a first region of a first CD137 molecules and a secondregion of a second molecule, wherein the first and/or second regioncomprises the amino acid sequence of SEQ ID NO: 34.

In certain embodiments, the antibody is a multivalent antibody and iscapable of binding to two or more molecules of CD137 simultaneously.

In certain embodiments, the antibody disclosed herein does notsubstantially inhibit human CD137 from binding to human CD137L.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD137 and does not substantiallyinhibit human CD137 from binding to human CD137L.

In certain embodiments, the antibody does not substantially inhibit asoluble fragment of human CD137 from binding to a soluble fragment ofhuman CD137L. In certain embodiments, the antibody does notsubstantially inhibit a CD137-expressing cell from binding to a solublefragment of human CD137L. In certain embodiments, the antibody does notsubstantially inhibit a CD137-expressing cell from binding to aCD137L-expressing cell.

In certain embodiments, the antibody does not inhibit a soluble fragmentof human CD137 from binding to a soluble fragment of human CD137L. Incertain embodiments, the antibody does not inhibit a CD137-expressingcell from binding to a soluble fragment of human CD137L. In certainembodiments, the antibody does not inhibit a CD137-expressing cell frombinding to a CD137L-expressing cell.

In certain embodiments, the antibody disclosed herein is agonistic tohuman CD137. In certain embodiments, the antibody increases or promotesan activity of human CD137. In certain embodiments, the ability of theantibody to increase or promote an activity of human CD137 is dependenton crosslinking of the antibody. In certain embodiments, the antibodydoes not substantially increase or promote an activity of human CD137 inthe absence of crosslinking.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to CD137 (e.g., human CD137) and increases orpromotes an activity of human CD137, wherein the ability of the antibodyto increase or promote an activity of human CD137 is dependent oncrosslinking of the antibody. In certain embodiments, the antibody doesnot substantially increase or promote an activity of human CD137 in theabsence of crosslinking.

In certain embodiments, the ability of the antibody to increase orpromote an activity of human CD137 is dependent on the presence ofCD137L.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to CD137 (e.g., human CD137) and increases orpromotes an activity of human CD137, wherein the ability of the antibodyto increase or promote an activity of human CD137 is dependent on thepresence of CD137L.

In certain embodiments, the ability of the antibody to increase orpromote an activity of human CD137 positively correlates with theconcentration of CD137L. In certain embodiments, the ability of theantibody to increase or promote the activity of human CD137 is asubstantially increasing function of the concentration of the CD137L. Incertain embodiments, the antibody does not substantially increase orpromote an activity of human CD137 in the absence of CD137L. In certainembodiments, the antibody does not increase or promote an activity ofhuman CD137 in the absence of CD137L.

In certain embodiments, the activity of human CD137 comprises activatinga T-cell expressing the human CD137. In certain embodiments, theactivity of human CD137 comprises inducing IL-2 production by peripheralblood mononuclear cells (PBMCs) stimulated with staphylococcalenterotoxin A (SEA). In certain embodiments, the activity of human CD137comprises activating a natural killer (NK) cell expressing the humanCD137. In certain embodiments, the activity of human CD137 comprisesactivating an antigen-presenting cell (APC) expressing CD137L.

In certain embodiments, the antibody binds to the same epitope of humanCD137 as an antibody comprising a VH comprising the amino acid sequenceof SEQ ID NO: 7 and a VL comprising the amino acid sequence of SEQ IDNO: 8.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD137, wherein the antibody binds tothe same epitope of human CD137 as an antibody comprising a VHcomprising the amino acid sequence of SEQ ID NO: 7 and a VL comprisingthe amino acid sequence of SEQ ID NO: 8. In certain embodiments, theantibody binds to an epitope located within a CRD4 domain of humanCD137. In certain embodiments, the CRD4 domain of human CD137 comprisesthe amino acid sequence set forth in SEQ ID NO: 42.

In certain embodiments, the antibody disclosed herein binds to anepitope located within a region of human CD137 consisting of the aminoacid sequence of any one of SEQ ID NOs: 26-31 and 43. In certainembodiments, the antibody binds to an epitope located within a region ofhuman CD137 consisting of the amino acid sequence of SEQ ID NO: 43.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD137, wherein the antibody binds to anepitope located within a region of human CD137 consisting of the aminoacid sequence of any one of SEQ ID NOs: 26-31 and 43. In certainembodiments, the antibody binds to an epitope located within a region ofhuman CD137 consisting of the amino acid sequence of SEQ ID NO: 43.

In certain embodiments, the antibody does not substantially bind to aprotein comprising the amino acid sequence of SEQ ID NO: 45. In certainembodiments, the antibody specifically binds to a protein comprising theamino acid sequence of SEQ ID NO: 46. In certain embodiments, theantibody specifically binds to a protein comprising the amino acidsequence of SEQ ID NO: 46, and does not substantially bind to a proteincomprising the amino acid sequence of SEQ ID NO: 45. In certainembodiments, the antibody does not substantially bind to a proteinconsisting of or consisting essentially of the amino acid sequence ofSEQ ID NO: 45. In certain embodiments, the antibody specifically bindsto a protein consisting of or consisting essentially of the amino acidsequence of SEQ ID NO: 46. In certain embodiments, the antibodyspecifically binds to a protein consisting of or consisting essentiallyof the amino acid sequence of SEQ ID NO: 46, and does not substantiallybind to a protein consisting of or consisting essentially of the aminoacid sequence of SEQ ID NO: 45.

In certain embodiments, the antibody comprises a VH and a VL, wherein:(a) an F(ab′)2 comprising two of each of the VH and the VL binds to anepitope located within a region of human CD137 consisting of the aminoacid sequence of SEQ ID NO: 27; and/or (b) a Fab comprising the VH andthe VL binds to an epitope located within a region of human CD137consisting of the amino acid sequence of SEQ ID NO: 26 and, optionally,an epitope located within a region of human CD137 consisting of theamino acid sequence of SEQ ID NO: 28 or 29.

In certain embodiments, the antibody comprises a VH and a VL, wherein:(a) if the antibody is formatted as a F(ab′)₂ comprising two of each ofthe VH and the VL, the F(ab′)₂ binds to an epitope located within aregion of human CD137 consisting of the amino acid sequence of SEQ IDNO: 27; and/or (b) if the antibody is formatted as a Fab comprising theVH and the VL, the Fab binds to an epitope located within a region ofhuman CD137 consisting of the amino acid sequence of SEQ ID NO: 26 andoptionally an epitope located within a region of human CD137 consistingof the amino acid sequence of SEQ ID NO: 28 or 29.

In certain embodiments, the antibody comprises a VH and a VL, wherein:(a) an F(ab′)₂ comprising two of each of the VH and the VL substantiallyreduces the exchange of hydrogen with deuterium in a region of CD137consisting of the amino acid sequence of SEQ ID NO: 34 relative to theexchange of hydrogen with deuterium in the same region in the absence ofthe F(ab′)₂, as measured by a hydrogen/deuterium exchange assay; and (b)a Fab comprising the VH and the VL does not substantially reduce theexchange of hydrogen with deuterium in a region of CD137 consisting ofthe amino acid sequence of SEQ ID NO: 34 relative to the exchange ofhydrogen with deuterium in the same region in the absence of the Fab, asmeasured by a hydrogen/deuterium exchange assay.

In certain embodiments, the antibody comprises a VH and a VL, wherein:(a) if the antibody is formatted as a F(ab′)₂ comprising two of each ofthe VH and the VL, the F(ab′)₂ substantially reduces the exchange ofhydrogen with deuterium in a region of CD137 consisting of the aminoacid sequence of SEQ ID NO: 34 relative to the exchange of hydrogen withdeuterium in the same region in the absence of the F(ab′)₂, as measuredby a hydrogen/deuterium exchange assay; and (b) if the antibody isformatted as a Fab comprising the VH and the VL, the Fab does notsubstantially reduces the exchange of hydrogen with deuterium in aregion of CD137 consisting of the amino acid sequence of SEQ ID NO: 34relative to the exchange of hydrogen with deuterium in the same regionin the absence of the Fab, as measured by a hydrogen/deuterium exchangeassay.

In certain embodiments, the antibody specifically binds to a proteincomprising the amino acid sequence of SEQ ID NO: 37, the antibody doesnot specifically bind to a protein comprising the amino acid sequence ofSEQ ID NO: 38.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD137, wherein the antibodyspecifically binds to a protein comprising the amino acid sequence ofSEQ ID NO: 37; and the antibody does not specifically bind to a proteincomprising the amino acid sequence of SEQ ID NO: 38.

In certain embodiments, the antibody disclosed herein is a humanantibody. In certain embodiments, the antibody disclosed herein is amultispecific antibody.

In certain embodiments, the antibody disclosed herein is conjugated to acytotoxic agent, cytostatic agent, toxin, radionuclide, or detectablelabel. In certain embodiments, the antibody is conjugated to a secondantibody.

In another aspect, the instant disclosure provides an isolatedpolynucleotide encoding a VH and/or a VL, or a heavy chain and/or alight chain of the antibody as disclosed herein. In another aspect, theinstant disclosure provides a vector comprising the polynucleotide asdisclosed herein. In another aspect, the instant disclosure provides arecombinant host cell comprising the polynucleotide or the vector asdisclosed herein.

In another aspect, the instant disclosure provides a pharmaceuticalcomposition comprising the antibody, polynucleotide, vector, or hostcell as disclosed herein; and a pharmaceutically acceptable carrier orexcipient.

In another aspect, the instant disclosure provides a method of producingan antibody that specifically binds to human CD137, the methodcomprising culturing the host cell as disclosed herein under suitableconditions so that the polynucleotide is expressed and the antibody isproduced.

In another aspect, the instant disclosure provides a method ofincreasing an immune response in a subject, the method comprisingadministering to the subject an effective amount of the antibody,polynucleotide, vector, host cell, or pharmaceutical composition asdisclosed herein.

In another aspect, the instant disclosure provides a method ofincreasing T cell activation and/or NK cell activation in response to anantigen in a subject, the method comprising administering to the subjectan effective amount of the antibody, polynucleotide, vector, host cell,or pharmaceutical composition as disclosed herein.

In another aspect, the instant disclosure provides a method of treatingcancer in a subject, the method comprising administering to the subjectan effective amount of the antibody, polynucleotide, vector, host cell,or pharmaceutical composition as disclosed herein.

In certain embodiments, the antibody, polynucleotide, vector, host cell,or pharmaceutical composition is administered systemically. In certainembodiments, the antibody, polynucleotide, vector, host cell, orpharmaceutical composition is administered intravenously. In certainembodiments, the antibody, polynucleotide, vector, host cell, orpharmaceutical composition is administered subcutaneously,intratumorally, or is delivered to a tumor draining lymph node.

In certain embodiments, the method of increasing an immune response in asubject, the method of increasing T cell activation and/or NK cellactivation in response to an antigen in a subject, or the method oftreating cancer in a subject disclosed herein further comprisesadministering an additional therapeutic agent to the subject. In certainembodiments, the additional therapeutic agent is a chemotherapeuticagent. In certain embodiments, the additional therapeutic agent is acheckpoint targeting agent. In certain embodiments, the checkpointtargeting agent is selected from the group consisting of an antagonistanti-PD-1 antibody, an antagonist anti-PD-L1 antibody, an antagonistanti-PD-L2 antibody, an antagonist anti-CTLA-4 antibody, an antagonistanti-TIM-3 antibody, an antagonist anti-LAG-3 antibody, an antagonistanti-VISTA antibody, an antagonist anti-CD96 antibody, an antagonistanti-CEACAM1 antibody, an antagonist anti-TIGIT antibody, an agonistanti-GITR antibody, and an agonist anti-OX40 antibody. In certainembodiments, the additional therapeutic agent is an anti-PD-1 antibody,optionally wherein the anti-PD-1 antibody is pembrolizumab or nivolumab.In certain embodiments, the additional therapeutic agent is an inhibitorof indoleamine-2,3-dioxygenase (IDO). In certain embodiments, theinhibitor is selected from the group consisting of epacadostat, F001287,indoximod, and NLG919. In certain embodiments, the additionaltherapeutic agent is a vaccine. In certain embodiments, the vaccinecomprises a heat shock protein peptide complex (HSPPC) comprising a heatshock protein complexed with an antigenic peptide. In certainembodiments, the heat shock protein is hsc70 and is complexed with atumor-associated antigenic peptide. In certain embodiments, the heatshock protein is gp96 protein and is complexed with a tumor-associatedantigenic peptide, wherein the HSPPC is derived from a tumor obtainedfrom a subject.

In another aspect, the instant disclosure provides a method of treatingan infectious disease in a subject, the method comprising administeringto the subject an effective amount of the antibody, polynucleotide,vector, host cell, or pharmaceutical composition as disclosed herein.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are a series of flow cytometry graphs showing thebinding of anti-CD137 antibody BA001 or an IgG1 isotype control antibodyto cells expressing human CD137 (FIG. 1A) or cynomolgus CD137 (FIG. 1B)on their cell surfaces. In FIG. 1A, binding to human CD137 was assessedfor Jurkat cells engineered to express human CD137 on their surfaces(left panel), activated human CEM/C1 T cells expressing endogenous CD137(middle panel), or activated human primary CD8+ T cells (right panel).In FIG. 1B, binding to cynomolgus CD137 was assessed for Jurkat cellsengineered to express cynomolgus CD137 on their surfaces (left panel) oractivated cynomolgus primary CD8+ T cells (right panel).

FIGS. 2A and 2B are surface plasmon resonance graphs showing binding ofhuman CD137L to human CD137, in the context of a CD137/BA001-F(ab′)₂complex. In FIG. 2A, BA001-F(ab′)₂ was bound to a flow cell, and thenCD137 was run over the flow cell, thereby forming a CD137/BA001-F(ab′)₂complex. CD137L was then run over the flow cell and was shown to bind tothe complex. In FIG. 2B, preformed CD137/BA001-F(ab′)₂ complex was firstbound to the flow cell. CD137L was then run over the flow cell and wasshown to bind to the complex.

FIGS. 3A-3C are graphs showing that anti-human CD137 antibody BA001 doesnot block CD137L binding to CD137. FIG. 3A is a series of flow cytometryplots showing that BA001 does not block binding of cells expressingCD137L on their surfaces to cells expressing CD137 on their surfaces.The top row of plots shows side scatter (SSC) and forward scatter (FSC)signal for each antibody, while the bottom row of plots showsJurkat-CD137 (PE) and Jurkat-CD137L (FITC) signal for each antibody.FIGS. 3B and 3C are graphs showing in a co-culture ofanti-CD137L-expressing cells and CD137-expressing cells, the percentageof conjugated cells out of the total number of cells, wherein ananti-CD137 antibody or isotype control antibody was added prior to (FIG.3B) or after (FIG. 3C) the two types of cells were combined in a mixedculture.

FIGS. 4A-4B are graphs showing crosslinking dependency of anti-CD137antibody BA001. FIG. 4A illustrates the NFκB-luciferase reporteractivities in Jurkat cells expressing human CD137 incubated with 2 μg/mLof crosslinked BA001, an isotype control, or reference anti-CD137antibody #2 in the absence of 1 μg/mL CD137L. The reporter activitiesare represented by luminescence levels, and are plotted against the logmolar ratio of the crosslinker (AffiniPure F(ab′)₂ Fragment GoatAnti-Human IgG) to antibody. FIG. 4B shows the NFκB-luciferase reporteractivities in Jurkat cells expressing human CD137 co-cultured withCD16-expressing CHO cells.

FIG. 5 is a graph showing the production of IL-2 induced by anti-CD137antibodies (i.e., BA001 and two reference anti-CD137 antibodies) orcorresponding isotype control antibodies (i.e., IgG1, IgG2, and IgG4isotype control antibodies, respectively) in human peripheral bloodmononuclear cells (PBMCs) upon Staphylococcal Enterotoxin A (SEA)stimulation.

FIGS. 6A-6C are graphs showing the production of IL-2 induced byanti-CD137 antibodies in purified human T cells stimulated with anti-CD3antibody (FIGS. 6A and 6B). CD137L expression was assessed in thepurified human T cells used in these experiments by flow cytometry. Nodetectable CD137L expression was observed on these cells (FIG. 6C).

FIG. 7A is a graph showing crosslinking- and ligand-dependency ofanti-CD137 antibody BA001 in the Jurkat reporter cells measured in thepresence or absence of 1 μg/mL CD137L. FIG. 7B is a histogram showingthe expression (or lack thereof) of CD137 and CD137L on the surfaces ofthe Jurkat reporter cells. Expression was analyzed from freshly thawedcells (“0 h”), cells cultured for 4 hours post thawing (“4 h”), andcells cultured for 24 hours post thawing (“24 h”).

FIGS. 8A-8D are graphs showing NFκB-luciferase reporter activity inJurkat cells expressing either human CD137 (FIGS. 8A and 8B) orcynomolgus CD137 (FIGS. 8C and 8D) and incubated with serial dilutionsof the anti-CD137 antibody BA001 or an isotype control antibody. In oneset of samples, cells were also incubated in the presence (FIGS. 8B and8D) or absence (FIGS. 8A and 8C) of human CD137L.

FIGS. 9A-9C are a series of graphs showing NFκB-luciferase reporteractivity in Jurkat cells expressing human CD137 and incubated with (i) 2μg/ml of anti-CD137 antibody (BA001 or one of two reference anti-CD137antibodies) or an appropriate isotype control antibody, and (ii) serialdilutions of human CD137L (ligand). In a first set of samples, theanti-CD137 antibody or isotype control antibody was added before CD137L(FIG. 9A). In a second set of samples, the anti-CD137 antibody orisotype control antibody was added simultaneously with CD137L (FIG. 9B).In a third set of samples, CD137L was added before the anti-CD137antibody or isotype control antibody (FIG. 9C).

FIG. 10A is a graph showing the production of IL-2 induced by Fcvariants of BA001 or corresponding isotype control antibodies in humanperipheral blood mononuclear cells (PBMCs) upon StaphylococcalEnterotoxin A (SEA) stimulation.

FIG. 10B is a graph showing production of IL-2 induced by serialdilutions of Fc variants of BA001 or corresponding isotype controlantibodies in human peripheral blood mononuclear cells (PBMCs) uponStaphylococcal Enterotoxin A (SEA) stimulation.

FIG. 11 is a series of NFκB-luciferase reporter activity in Jurkat cellsexpressing either human or cynomolgus CD137 and incubated with serialdilutions of an Fc variant of BA001 or an appropriate isotype controlantibody. In one set of samples, cells were also incubated in thepresence (right column) or absence (left column) of human CD137L.

FIGS. 12A and 12B are graphs showing production of IL-2 induced byantibodies in human peripheral blood mononuclear cells (PBMCs) uponStaphylococcal Enterotoxin A (SEA) stimulation. Antibodies tested inFIG. 12A include anti-CD137 antibody BA001, isotype control antibody, ananti-PD-1 antibody, and a combination of BA001 and the anti-PD-1antibody. Antibodies tested in FIG. 12B include anti-CD137 antibodyBA001, isotype control antibody, an anti-OX40 antibody, and acombination of BA001 and the anti-OX40 antibody.

FIG. 13 is a sequence alignment for human CD137 and cynomolgus monkeyCD137. An “*” (asterisk) indicates positions which have a single, fullyconserved residue. A “:” (colon) indicates conservation between groupsof strongly similar properties. A “.” (period) indicates conservationbetween groups of weakly similar properties. The region boxed withdotted lines (DPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCD, SEQ ID NO: 34)exhibited a mild decrease in deuterium uptake when human CD137 was boundto BA001-F(ab′)₂, possibly owing to CD137 homodimerization at thisregion. The region boxed with solid lines (FNDQKRGICRPWTNCSL, SEQ ID NO:26) exhibited a strong decrease in deuterium uptake when human CD137 wasbound to BA001-Fab.

FIGS. 14A and 14B are a series of diagrams showing epitope mapping ofBA001 by FACS. In FIG. 14A, a series of human-to-murine sequence switchmutants of CD137 were generated for each of the regions shown (i.e.,5014, 5015, 5016, 5017, and 5018, see Table 5 below). These mutantconstructs were then transfected into Jurkat cells for analysis ofanti-CD137 antibody binding by FACS. FIG. 14B shows cell binding datafor BA001, reference anti-CD137 antibodies #1 and #2 (“Reference #1” and“Reference #2,” respectively), and isotype control antibody toengineered Jurkat cells expressing each of the switch mutants describedabove.

FIGS. 15A-15C show fine mapping of CD137 epitope by surface plasmonresonance (SPR) assay. FIG. 15A is a sequence alignment for human CD137and murine CD137. An “*” (asterisk) indicates positions which have asingle, fully conserved residue. A “:” (colon) indicates conservationbetween groups of strongly similar properties. A “.” (period) indicatesconservation between groups of weakly similar properties. The regionboxed with solid lines (FNDQKRGICRPWTNCSL, SEQ ID NO: 26) is an epitoperegion identified by the hydrogen/deuterium exchange assay asillustrated in FIG. 13. The region boxed with dotted lines(LTKKGCKDCCFGTFNDQKRGICRPWTNC, SEQ ID NO: 30) is the 5017 regionidentified from the binding assay using the human-mouse fusionconstructs as illustrated in FIGS. 14A and 14B. The region highlightedby a solid line (KRGI, SEQ ID NO: 43) indicates the amino acid sequencethat has been switched between the human and murine CD137 to generatechimeric proteins. FIG. 15B is a sensorgram showing the binding of BA001to human CD137 and the chimeric proteins “human to mouse” and “mouse tohuman” by SPR assay. FIG. 15C is a sensorgram showing the binding ofreference anti-CD137 antibody #1 (“Reference #1”) to the same CD137proteins in a similar SPR assay.

FIGS. 16A-16D are a series of graphs showing the binding of four BA001variants, BA049, BA050, BA051, and BA052, to the extracellular domainsof human CD137 (FIG. 16A), cynomolgus CD137 (FIG. 16B), mouse-humanfusion construct 5017 (“mCD137-human112-139”) (FIG. 16C), andmouse-human fusion construct 5015 (“mCD137-human53-80”) (FIG. 16D), asmeasured by enzyme-linked immunosorbent assay (ELISA) using afluorescent label as readout. The median fluorescence intensity levelswere plotted against the concentrations of the anti-CD137 antibodies.

5. DETAILED DESCRIPTION

The instant disclosure provides antibodies that specifically bind toCD137 (e.g., human CD137 or cynomolgus CD137) and increase or promoteCD137 function, e.g., CD137-mediated immune activation. Also providedare pharmaceutical compositions comprising these antibodies, nucleicacids encoding these antibodies, expression vectors and host cells formaking these antibodies, and methods of treating a subject using theseantibodies. The antibodies disclosed herein are particularly useful forincreasing T cell activation in response to an antigen (e.g., a tumorantigen or an infectious disease antigen), and hence for treating cancerin a subject or treating or preventing an infectious disease in asubject. All instances of “isolated antibodies” described herein areadditionally contemplated as antibodies that may be, but need not be,isolated. All instances of “isolated polynucleotides” described hereinare additionally contemplated as polynucleotides that may be, but neednot be, isolated. All instances of “antibodies” described herein areadditionally contemplated as antibodies that may be, but need not be,isolated. All instances of “polynucleotides” described herein areadditionally contemplated as polynucleotides that may be, but need notbe, isolated.

5.1 Definitions

As used herein, the terms “about” and “approximately,” when used tomodify a numeric value or numeric range, indicate that deviations of 5%to 10% above (e.g., up to 5% to 10% above) and 5% to 10% below (e.g., upto 5% to 10% below) the value or range remain within the intendedmeaning of the recited value or range.

As used herein, the term “CD137” refers to TNF receptor superfamilymember 9 (also known as 4-1BB) that in humans is encoded by the TNFRSF9gene. As used herein, the term “human CD137” refers to a CD137 proteinencoded by a wild-type human CD137 gene (e.g., GenBank™ accession numberNM_001561.5) or an extracellular domain of such a protein. An exemplaryamino acid sequence of an immature human CD137 protein is provided asSEQ ID NO: 25. An exemplary amino acid sequence of a mature human CD137protein is provided as SEQ ID NO: 33. An exemplary amino acid sequenceof an extracellular domain of a mature human CD137 protein is providedas SEQ ID NO: 24.

As used herein, the terms “antibody” and “antibodies” include fulllength antibodies, antigen-binding fragments of full length antibodies,and molecules comprising antibody CDRs, VH regions, and/or VL regions.Examples of antibodies include, without limitation, monoclonalantibodies, recombinantly produced antibodies, monospecific antibodies,multispecific antibodies (including bispecific antibodies), humanantibodies, humanized antibodies, chimeric antibodies, immunoglobulins,synthetic antibodies, tetrameric antibodies comprising two heavy chainand two light chain molecules, an antibody light chain monomer, anantibody heavy chain monomer, an antibody light chain dimer, an antibodyheavy chain dimer, an antibody light chain-antibody heavy chain pair,intrabodies, heteroconjugate antibodies, antibody-drug conjugates,single domain antibodies, monovalent antibodies, single chain antibodiesor single-chain Fvs (scFv), camelized antibodies, affybodies, Fabfragments, F(ab′)₂ fragments, disulfide-linked Fvs (sdFv),anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Idantibodies), and antigen-binding fragments of any of the above. Incertain embodiments, antibodies described herein refer to polyclonalantibody populations. Antibodies can be of any type (e.g., IgG, IgE,IgM, IgD, IgA or IgY), any class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ orIgA₂), or any subclass (e.g., IgG_(2a) or IgG_(2b)) of immunoglobulinmolecule. In certain embodiments, antibodies described herein are IgGantibodies, or a class (e.g., human IgG₁ or IgG₄) or subclass thereof.In a specific embodiment, the antibody is a humanized monoclonalantibody. In another specific embodiment, the antibody is a humanmonoclonal antibody.

As used herein, the terms “VH region” and “VL region” refer,respectively, to single antibody heavy and light chain variable regions,comprising FR (Framework Regions) 1, 2, 3 and 4 and CDR (ComplementarityDetermining Regions) 1, 2 and 3 (see Kabat et al., (1991) Sequences ofProteins of Immunological Interest (NIH Publication No. 91-3242,Bethesda), which is herein incorporated by reference in its entirety).

As used herein, the term “CDR” or “complementarity determining region”means the noncontiguous antigen combining sites found within thevariable region of both heavy and light chain polypeptides. Theseparticular regions have been described by Kabat et al., J. Biol. Chem.252, 6609-6616 (1977) and Kabat et al., Sequences of protein ofimmunological interest. (1991), by Chothia et al., J. Mol. Biol.196:901-917 (1987), and by MacCallum et al., J. Mol. Biol. 262:732-745(1996), all of which are herein incorporated by reference in theirentireties, where the definitions include overlapping or subsets ofamino acid residues when compared against each other. In certainembodiments, the term “CDR” is a CDR as defined by MacCallum et al., J.Mol. Biol. 262:732-745 (1996) and Martin A. “Protein Sequence andStructure Analysis of Antibody Variable Domains,” in AntibodyEngineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439,Springer-Verlag, Berlin (2001). In certain embodiments, the term “CDR”is a CDR as defined by Kabat et al., J. Biol. Chem. 252, 6609-6616(1977) and Kabat et al., Sequences of protein of immunological interest.(1991). In certain embodiments, heavy chain CDRs and light chain CDRs ofan antibody are defined using different conventions. For example, incertain embodiments, the heavy chain CDRs are defined according toMacCallum (supra), and the light CDRs are defined according to Kabat(supra). CDRH1, CDRH2 and CDRH3 denote the heavy chain CDRs, and CDRL1,CDRL2 and CDRL3 denote the light chain CDRs.

As used herein, the term “framework (FR) amino acid residues” refers tothose amino acids in the framework region of an immunoglobulin chain.The term “framework region” or “FR region” as used herein, includes theamino acid residues that are part of the variable region, but are notpart of the CDRs (e.g., using the Kabat or MacCallum definition ofCDRs).

As used herein, the terms “variable region” and “variable domain” areused interchangeably and are common in the art. The variable regiontypically refers to a portion of an antibody, generally, a portion of alight or heavy chain, typically about the amino-terminal 110 to 120amino acids or 110 to 125 amino acids in the mature heavy chain andabout 90 to 115 amino acids in the mature light chain, which differextensively in sequence among antibodies and are used in the binding andspecificity of a particular antibody for its particular antigen. Thevariability in sequence is concentrated in those regions calledcomplementarity determining regions (CDRs) while the more highlyconserved regions in the variable domain are called framework regions(FR). Without wishing to be bound by any particular mechanism or theory,it is believed that the CDRs of the light and heavy chains are primarilyresponsible for the interaction and specificity of the antibody withantigen. In certain embodiments, the variable region is a human variableregion. In certain embodiments, the variable region comprises rodent ormurine CDRs and human framework regions (FRs). In particularembodiments, the variable region is a primate (e.g., non-human primate)variable region. In certain embodiments, the variable region comprisesrodent or murine CDRs and primate (e.g., non-human primate) frameworkregions (FRs).

The terms “VL” and “VL domain” are used interchangeably to refer to thelight chain variable region of an antibody.

The terms “VH” and “VH domain” are used interchangeably to refer to theheavy chain variable region of an antibody.

As used herein, the terms “constant region” and “constant domain” areinterchangeable and are common in the art. The constant region is anantibody portion, e.g., a carboxyl terminal portion of a light and/orheavy chain which is not directly involved in binding of an antibody toantigen but which can exhibit various effector functions, such asinteraction with an Fc receptor (e.g., Fc gamma receptor). The constantregion of an immunoglobulin molecule generally has a more conservedamino acid sequence relative to an immunoglobulin variable domain.

As used herein, the term “heavy chain” when used in reference to anantibody can refer to any distinct type, e.g., alpha (α), delta (δ),epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence ofthe constant domain, which give rise to IgA, IgD, IgE, IgG, and IgMclasses of antibodies, respectively, including subclasses of IgG, e.g.,IgG₁, IgG₂, IgG₃, and IgG₄.

As used herein, the term “light chain” when used in reference to anantibody can refer to any distinct type, e.g., kappa (κ) or lambda (λ)based on the amino acid sequence of the constant domains. Light chainamino acid sequences are well known in the art. In specific embodiments,the light chain is a human light chain.

As used herein, the term “EU numbering system” refers to the EUnumbering convention for the constant regions of an antibody, asdescribed in Edelman, G. M. et al., Proc. Natl. Acad. USA, 63, 78-85(1969) and Kabat et al, Sequences of Proteins of Immunological Interest,U.S. Dept. Health and Human Services, 5th edition, 1991, each of whichis herein incorporated by reference in its entirety.

“Binding affinity” generally refers to the strength of the sum total ofnon-covalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (K_(D)). Affinity can be measured and/or expressedin a number of ways known in the art, including, but not limited to,equilibrium dissociation constant (K_(D)), and equilibrium associationconstant (K_(A)). The K_(D) is calculated from the quotient ofk_(off)/k_(on), whereas K_(A) is calculated from the quotient ofk_(on)/k_(off). k_(on) refers to the association rate constant of, e.g.,an antibody to an antigen, and k_(off) refers to the dissociation rateconstant of, e.g., an antibody to an antigen. The k_(on) and k_(off) canbe determined by techniques known to one of ordinary skill in the art,such as BIAcore® or KinExA. As used herein, a “lower affinity” refers toa larger K_(D).

As used herein, the terms “specifically binds,” “specificallyrecognizes,” “immunospecifically binds,” and “immunospecificallyrecognizes” are analogous terms in the context of antibodies and referto molecules that bind to an antigen (e.g., epitope or immune complex)as such binding is understood by one skilled in the art. For example, amolecule that specifically binds to an antigen can bind to otherpeptides or polypeptides, generally with lower affinity as determinedby, e.g., immunoassays, BIAcore®, KinExA 3000 instrument (SapidyneInstruments, Boise, Id.), or other assays known in the art. In aspecific embodiment, molecules that specifically bind to an antigen bindto the antigen with a K_(A) that is at least 2 logs (e.g., factors of10), 2.5 logs, 3 logs, 4 logs or greater than the K_(A) when themolecules bind non-specifically to another antigen.

In another specific embodiment, molecules that specifically bind to anantigen do not cross react with other proteins under similar bindingconditions. In another specific embodiment, molecules that specificallybind to CD137 do not cross react with other non-CD137 proteins. In aspecific embodiment, provided herein is an antibody that binds to CD137(e.g., human CD137 or cynomolgus CD137) with higher affinity than toanother unrelated antigen. In certain embodiments, provided herein is anantibody that binds to CD137 (e.g., human CD137 or cynomolgus CD137)with a 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or higher affinity than to another, unrelated antigen asmeasured by, e.g., a radioimmunoassay, surface plasmon resonance, orkinetic exclusion assay. In a specific embodiment, the extent of bindingof an anti-CD137 antibody described herein to an unrelated, non-CD137protein is less than 10%, 15%, or 20% of the binding of the antibody toCD137 protein as measured by, e.g., a radioimmunoassay.

As used herein, A “does not substantially inhibit” B means that B is notreduced by more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% inthe presence of A relative to B in the absence of A.

As used herein, B is a “substantially increasing function” of A over aspecified domain of A values if B substantially increases as A increasesover the specified domain, e.g., in a given experiment, or using meanvalues from multiple experiments. This definition allows for a value ofB corresponding to a specified value of A to be up to 1%, 2%, 3%, 4%,5%, 10%, 15%), or 20%) lower relative to a value of B corresponding toany lower value of A.

As used herein, an “epitope” is a term in the art and refers to alocalized region of an antigen to which an antibody can specificallybind. An epitope can be, for example, contiguous amino acids of apolypeptide (linear or contiguous epitope) or an epitope can, forexample, come together from two or more non-contiguous regions of apolypeptide or polypeptides (conformational, non-linear, discontinuous,or non-contiguous epitope). In certain embodiments, the epitope to whichan antibody binds can be determined by, e.g., NMR spectroscopy, X-raydiffraction crystallography studies, ELISA assays, hydrogen/deuteriumexchange coupled with mass spectrometry (e.g., liquid chromatographyelectrospray mass spectrometry), array-based oligo-peptide scanningassays (e.g., constraining peptides using CLIPS (Chemical Linkage ofPeptides onto Scaffolds) to map discontinuous or conformationalepitopes), and/or mutagenesis mapping (e.g., site-directed mutagenesismapping). For X-ray crystallography, crystallization may be accomplishedusing any of the known methods in the art (e.g., Giegé R et al., (1994)Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A(1990) Eur J Biochem 189: 1-23; Chayen N E (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, each of whichis herein incorporated by reference in its entirety). Antibody:antigencrystals may be studied using well known X-ray diffraction techniquesand may be refined using computer software such as X-PLOR (YaleUniversity, 1992, distributed by Molecular Simulations, Inc.; see, e.g.,Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff H W et al.; U.S.2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D BiolCrystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter C W; Roversi P et al., (2000) Acta Crystallogr D BiolCrystallogr 56(Pt 10): 1316-1323), each of which is herein incorporatedby reference in its entirety. Mutagenesis mapping studies may beaccomplished using any method known to one of skill in the art. See,e.g., Champe M et al., (1995) J Biol Chem 270: 1388-1394 and CunninghamB C & Wells J A (1989) Science 244: 1081-1085, each of which is hereinincorporated by reference in its entirety, for a description ofmutagenesis techniques, including alanine scanning mutagenesistechniques. CLIPS (Chemical Linkage of Peptides onto Scaffolds) is atechnology to present one or more peptides in a structurally constrainedconfiguration to behave as functional mimics of complex protein domains.See, e.g., U.S. Publication Nos. US 2008/0139407 A1 and US 2007/099240A1, and U.S. Pat. No. 7,972,993, each of which is herein incorporated byreference in its entirety. In a specific embodiment, the epitope of anantibody is determined using alanine scanning mutagenesis studies. In aspecific embodiment, the epitope of an antibody is determined usinghydrogen/deuterium exchange coupled with mass spectrometry. In aspecific embodiment, the epitope of an antibody is determined usingCLIPS Epitope Mapping Technology from Pepscan Therapeutics. In aspecific embodiment, the epitope of an antibody is determined by proteinmutagenesis, e.g., by generating switch mutants of an antigen withportions of its ortholog from another species and then testing theswitch mutants for loss of antibody binding (e.g., by a FACS-based cellbinding assay, as described herein).

As used herein, the term “an epitope located within” a region of humanCD137 refers to an epitope comprising one or more of the amino acidresidues of the specified region. In certain embodiments, the epitopecomprises each one of the amino acid residues located within thespecified region. In certain embodiments, the epitope consists of eachone of the amino acid residues located within the specified region. Incertain embodiments, one or more additional amino acid residues of humanCD137 outside the specified region bind to an antibody together with anepitope located within the specified region.

As used herein, the terms “T cell receptor” and “TCR” are usedinterchangeably and refer to full length heterodimeric αβ or γδ TCRs,antigen-binding fragments of full length TCRs, and molecules comprisingTCR CDRs or variable regions. Examples of TCRs include, but are notlimited to, full length TCRs, antigen-binding fragments of full lengthTCRs, soluble TCRs lacking transmembrane and cytoplasmic regions,single-chain TCRs containing variable regions of TCRs attached by aflexible linker, TCR chains linked by an engineered disulfide bond,monospecific TCRs, multi-specific TCRs (including bispecific TCRs), TCRfusions, human TCRs, humanized TCRs, chimeric TCRs, recombinantlyproduced TCRs, and synthetic TCRs. The term encompasses wild-type TCRsand genetically engineered TCRs (e.g., a chimeric TCR comprising achimeric TCR chain which includes a first portion from a TCR of a firstspecies and a second portion from a TCR of a second species).

As used herein, the term “level of CD137 multimerization” refers to therelative amount of multimeric (e.g., dimeric) CD137 compared tomonomeric CD137 in a population of CD137 molecules (e.g., a populationof CD137 molecules expressed on the surface of one or more cells).

As used herein, the terms “major histocompatibility complex” and “MHC”are used interchangeably and refer to an MHC class I molecule and/or anMHC class II molecule.

As used herein, the term “peptide-MHC complex” refers to an MHC molecule(MHC class I or MHC class II) with a peptide bound in the art-recognizedpeptide binding pocket of the MHC.

As used herein, the term “treat,” “treating,” and “treatment” refer totherapeutic or preventative measures described herein. The methods of“treatment” employ administration of an antibody to a subject having adisease or disorder, or predisposed to having such a disease ordisorder, in order to prevent, cure, delay, reduce the severity of, orameliorate one or more symptoms of the disease or disorder or recurringdisease or disorder, or in order to prolong the survival of a subjectbeyond that expected in the absence of such treatment.

As used herein, the term “effective amount” in the context of theadministration of a therapy to a subject refers to the amount of atherapy that achieves a desired prophylactic or therapeutic effect.

As used herein, the term “subject” includes any human or non-humananimal. In one embodiment, the subject is a human or non-human mammal.In one embodiment, the subject is a human.

The determination of “percent identity” between two sequences (e.g.,amino acid sequences or nucleic acid sequences) can be accomplishedusing a mathematical algorithm. A specific, non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin S & Altschul S F (1990) PNAS 87: 2264-2268,modified as in Karlin S & Altschul S F (1993) PNAS 90: 5873-5877, eachof which is herein incorporated by reference in its entirety. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul S F et al., (1990) J Mol Biol 215: 403, which is hereinincorporated by reference in its entirety. BLAST nucleotide searches canbe performed with the NBLAST nucleotide program parameters set, e.g.,for score=100, wordlength=12 to obtain nucleotide sequences homologousto a nucleic acid molecules described herein. BLAST protein searches canbe performed with the XBLAST program parameters set, e.g., to score 50,wordlength=3 to obtain amino acid sequences homologous to a proteinmolecule described herein. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul S F etal., (1997) Nuc Acids Res 25: 3389-3402, which is herein incorporated byreference in its entirety. Alternatively, PSI BLAST can be used toperform an iterated search which detects distant relationships betweenmolecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blastprograms, the default parameters of the respective programs (e.g., of)(BLAST and NBLAST) can be used (see, e.g., National Center forBiotechnology Information (NCBI) on the worldwide web,ncbi.nlm.nih.gov). Another specific, non-limiting example of amathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is hereinincorporated by reference in its entirety. Such an algorithm isincorporated in the ALIGN program (version 2.0) which is part of the GCGsequence alignment software package. When utilizing the ALIGN programfor comparing amino acid sequences, a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

5.2 Anti-CD137 Antibodies

In one aspect the instant disclosure provides antibodies thatspecifically bind to CD137 (e.g., human CD137 or cynomolgus CD137) andincrease or promote CD137 function. The amino acid sequences ofexemplary antibodies are set forth in Table 1, herein.

TABLE 1 Amino acid sequences of exemplary anti-CD137 antibodies. SEQ IDDescription Amino Acid Sequence NO: CDRH1 consensus X₁X₂X₃X₄H, wherein82 sequence 1 X₁ is G, A, D, E, L, N, Q, R, S, or W;X₂ is Y, F, H, N, R, or S; X₃ is Y or H; and X₄ is M, I, T, or VCDRH3 consensus X₁PX₂YX₃GX₄GLX₅X₆, wherein 83 sequence 1 X₁ is E or G;X₂ is G, A, R, or S; X₃ is Y, F, H, or S; X₄ is S, A, or T;X₅ is D or G; and X₆ is Y or H CDRL3 consensusQX₁WX₂X₃X₄X₅X₆X₇PGV, wherein 84 sequence 1 X₁ is V or I;X₂ is D, A, E, G, H, N, or Y; X₃ is S, A, E, F, L, P, R, T, W, or Y;X₄ is S, A, L, M, or R; X₅ is S, A, F, G, L, P, Q, R, or T;X₆ is D, E, H, V, or Y; and X₇ is H or Y CDRH1 consensusX₁X₂YX₃H, wherein 85 sequence 2 X₁ is G, A, D, L, R, S, or W;X₂ is Y, F, H, or N; and X₃ is M or V CDRH3 consensusEPGYX₁GX₂GLDX₃, wherein 86 sequence 2 X₁ is Y or F; X₂ is S or T; andX₃ is Y or H CDRL3 consensus QVWX₁X₂X₃X₄X₅X₆PGV, wherein 87 sequence 2X₁ is D, A, E, H, N, or Y; X₂ is S, A, E, L, R, or T;X₃ is S, A, L, or R; X₄ is S, A, F, G, L, P, Q, or R;X₅ is D, E, or V; and X₆ is H or Y BA001 CDRH1 GYYMH  1 BA001 CDRH2WINPNSGGTNYAQKFQG  2 BA001 CDRH3 EPGYYGSGLDY  3 BA001 CDRL1 GGDDIGDKRVH 4 BA001 CDRL2 EDRYRPS  5 BA001 CDRL3 QVWDSSSDHPGV  6 BA001 VHXVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW  7VRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLDYWGQGTLVTVSS, wherein X = glutamine (Q) or pyroglutamate (pE) BA001 VLSYVLTQPPSVSVAPGETARITCGGDDIGDKRVHWYQK  8KPDQAPVLVVYEDRYRPSGIPERISGSNSGNTATLTLSRVEAGDEADYYCQVWDSSSDHPGVFGGGTQLIIL BA001 full lengthXVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW  9 heavy chain (IgG1)VRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMT (without C-RDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLD terminal lysine)YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, wherein X = glutamine (Q) or pyroglutamate (pE) BA001 full lengthXVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 49 heavy chain (IgG1)VRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMT (with C-terminalRDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLD lysine)YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein X = glutamine (Q) or pyroglutamate (pE) BA001 IgG1XVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 10 N297A variant fullVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMT length heavy chainRDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLD (without C-YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG terminal lysine)CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, wherein X = glutamine (Q) or pyroglutamate (pE) BA001 IgG1XVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 50 N297A variant fullVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMT length heavy chainRDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLD (with C-terminalYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG lysine)CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein X = glutamine (Q) or pyroglutamate (pE) BA001 IgG1XVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 11 S267E L328FVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMT variant full lengthRDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLD heavy chainYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG (without C-CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS terminal lysine)LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, wherein X = glutamine (Q) or pyroglutamate (pE) BA001 IgG1XVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 51 S267E L328FVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMT variant full lengthRDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLD heavy chain (withYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG C-terminal lysine)CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein X = glutamine (Q) or pyroglutamate (pE) BA001 IgG2XVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 12 variant full lengthVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMT heavy chainRDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLD (without C-YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALG terminal lysine)CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,wherein X = glutamine (Q) or pyroglutamate (pE) BA001 IgG2XVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 52 variant full lengthVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMT heavy chain (withRDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLD C-terminal lysine)YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein X = glutamine (Q) or pyroglutamate (pE) BA001 IgG2XVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 13 N297A variant fullVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMT length heavy chainRDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLD (without C-YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALG terminal lysine)CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDINVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,wherein X = glutamine (Q) or pyroglutamate (pE) BA001 IgG2XVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 53 N297A variant fullVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMT length heavy chainRDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLD (with C-terminalYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALG lysine)CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDINVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein X = glutamine (Q) or pyroglutamate (pE) BA001 IgG4XVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 14 S228P variant fullVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMT length heavy chainRDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLD (without C-YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALG terminal lysine)CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG,wherein X = glutamine (Q) or pyroglutamate (pE) BA001 IgG4XVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 54 S228P variant fullVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMT length heavy chainRDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLD (with C-terminalYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALG lysine)CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK,wherein X = glutamine (Q) or pyroglutamate (pE) BA001 constantASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT 15 region (IgG1)VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGBA001 IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT 16 N297A variantVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL constant regionGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFScSVMHEALHNHYTQKSLSLSPGBA001 IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT 17 S267E L328FVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL variant constantGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA regionPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGBA001 IgG2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV 18 variant constantSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFG regionTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGBA001 IgG2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV 19 N297A variantSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFG constant regionTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDINVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGBA001 IgG4 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV 20 s228P variantSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG constant regionTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSLGBA001 full length SYVLTQPPSVSVAPGETARITCGGDDIGDKRVHWYQK 21 light chainKPDQAPVLVVYEDRYRPSGIPERISGSNSGNTATLTLSRVEAGDEADYYCQVWDSSSDHPGVFGGGTQUILGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQW KSHRSYSCQVTHEGSTVEKTVAPTECSBA001 light chain GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVT 22constant region VAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS BA001 scFvQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 55VRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLDYWGQGTLVTVSSGGGGSGGGGSGGGGASSYVLTQPPSVSVAPGETARITCGGDDIGDKRVHWYQKKPDQAPVLVVYEDRYRPSGIPERISGSNSGNTATLTLSRVEAGDEAD YYCQVWDSSSDHPGVFGGGTQLIILBA001 CDRH1 TFTGYYMH 56 (bold) plus N- terminal flanking residuesBA050 CDRH1 SFTGYYMH 57 (bold) plus N- terminal flanking residuesBA052 CDRH1 NFS GYYMH 58 (bold) plus N- terminal flanking residuesBA049 CDRH3 EPGYYGTGLDY 59 BA050 CDRL3 QVWNSSSDHPGV 60 BA051 CDRL3QVWDSSSDYPGV 61 BA052 CDRL3 QVWYSSPDHPGV 62 BA049 VHXVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 63VRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREPGYYGTGLDYWGQGTLVTVSS, wherein X = glutamine (Q) or pyroglutamate (pE) BA050 VHXVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYMHW 64VRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLDYWGQGTLVTVSS, wherein X = glutamine (Q) or pyroglutamate (pE) BA052 VHXVQLVQSGAEVKKPGASVKVSCKASGYNFSGYYMHW 65VRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTITRDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLDYWGQGTLVTVSS, wherein X = glutamine (Q) or pyroglutamate (pE) BA050 VLSYVLTQPPSVSVAPGETARITCGGDDIGDKRVHWYQK 66KPDQAPVLVVYEDRYRPSGIPERISGSNSGNTATLTLSRVEAGDEADYYCQVWNSSSDHPGVFGGGTQLIIL BA051 VLSYVLTQPPSVSVAPGETARITCGGDDIGDKRVHWYQK 67KPDQAPVLVVYEDRYRPSGIPERISGSNSGNTATLTLSRVEAGDEADYYCQVWDSSSDYPGVFGGGTQLIIL BA052 VLSYVLTQPPSVSVAPGETARITCGGDDIGDKRVHWYQK 68KPDQAPVLVVYEDRYRPSGIPERISGSNSGNTATLTLSRVEAGDEADYYCQVWYSSPDHPGVFGGGTQLIIL BA049 scFvXVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 69VRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREPGYYGTGLDYWGQGTLVTVSSGGGGSGGGGSGGGGASSYVLTQPPSVSVAPGETARITCGGDDIGDKRVHWYQKKPDQAPVLVVYEDRYRPSGIPERISGSNSGNTATLTLSRVEAGDEADYYCQVWDSSSDHPGVFGGGTQLIIL, wherein X =glutamine (Q) or pyroglutamate (pE) BA050 scFvXVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYMHW 70VRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLDYWGQGTLVTVSSGGGGSGGGGSGGGGASSYVLTQPPSVSVAPGETARITCGGDDIGDKRVHWYQKKPDQAPVLVVYEDRYRPSGIPERISGSNSGNTATLTLSRVEAGDEADYYCQVWNSSSDHPGVFGGGTQLIIL, wherein X =glutamine (Q) or pyroglutamate (pE) BA051 scFvXVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 71VRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLDYWGQGTLVTVSSGGGGSGGGGSGGGGASSYVLTQPPSVSVAPGETARITCGGDDIGDKRVHWYQKKPDQAPVLVVYEDRYRPSGIPERISGSNSGNTATLTLSRVEAGDEADYYCQVWDSSSDYPGVFGGGTQLIIL, wherein X =glutamine (Q) or pyroglutamate (pE) BA052 scFvXVQLVQSGAEVKKPGASVKVSCKASGYNFSGYYMHW 72VRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTITRDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLDYWGQGTLVTVSSGGGGSGGGGSGGGGASSYVLTQPPSVSVAPGETARITCGGDDIGDKRVHWYQKKPDQAPVLVVYEDRYRPSGIPERISGSNSGNTATLTLSRVEAGDEADYYCQVWYSSPDHPGVFGGGTQLIIL, wherein X =glutamine (Q) or pyroglutamate (pE) BA049 full-lengthXVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 73 heavy chainVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMT (without C-RDTSISTAYMELSRLRSDDTAVYYCAREPGYYGTGLD terminal lysine)YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, wherein X = glutamine (Q) or pyroglutamate (pE) BA049 full-lengthXVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 74 heavy chain (withVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMT C-terminal lysine)RDTSISTAYMELSRLRSDDTAVYYCAREPGYYGTGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein X = glutamine (Q) or pyroglutamate (pE) BA050 full-lengthXVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYMHW 75 heavy chainVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMT (without C-RDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLD terminal lysine)YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, wherein X = glutamine (Q) or pyroglutamate (pE) BA050 full-lengthXVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYMHW 76 heavy chain (withVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMT C-terminal lysine)RDTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein X = glutamine (Q) or pyroglutamate (pE) BA052 full-lengthXVQLVQSGAEVKKPGASVKVSCKASGYNFSGYYMHW 77 heavy chainVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTITR (without C-DTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLDY terminal lysine)WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, wherein X = glutamine (Q) or pyroglutamate (pE) BA052 full-lengthXVQLVQSGAEVKKPGASVKVSCKASGYNFSGYYMHW 78 heavy chain (withVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTITR C-terminal lysine)DTSISTAYMELSRLRSDDTAVYYCAREPGYYGSGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein X = glutamine (Q) or pyroglutamate (pE) BA050 full-lengthSYVLTQPPSVSVAPGETARITCGGDDIGDKRVHWYQK 79 light chainKPDQAPVLVVYEDRYRPSGIPERISGSNSGNTATLTLSRVEAGDEADYYCQVWNSSSDHPGVEGGGTQLIILGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQW KSHRSYSCQVTHEGSTVEKTVAPTECSBA051 full-length SYVLTQPPSVSVAPGETARITCGGDDIGDKRVHWYQK 80 light chainKPDQAPVLVVYEDRYRPSGIPERISGSNSGNTATLTLSRVEAGDEADYYCQVWDSSSDYPGVEGGGTQLIILGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQW KSHRSYSCQVTHEGSTVEKTVAPTECSBA052 full-length SYVLTQPPSVSVAPGETARITCGGDDIGDKRVHWYQK 81 light chainKPDQAPVLVVYEDRYRPSGIPERISGSNSGNTATLTLSRVEAGDEADYYCQVWYSSPDHPGVEGGGTQLIILGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQW KSHRSYSCQVTHEGSTVEKTVAPTECS

TABLE 2 VH, VL, scFv, and full length heavy and light chain sequences ofexemplary anti-CD137 antibodies. SEQ ID NO Full-length Full-lengthAntibody VH VL scFv heavy chain light chain BA001 7 8 55 9 21 BA049 63 869 74 21 BA050 64 66 70 76 79 BA051 7 67 71 49 80 BA052 65 68 72 78 81

TABLE 3 Closest germline genes to BA001. SEQ Closest ID germline geneAmino Acid Sequence NO: IGHV1-2*02QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVR 40 heavy chainQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSIS variable regionTAYMELSRLRSDDTAVYYCAR IGLV3-21*02SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPG 41 light chainQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGD variable regionEADYYCQVWDSSSDH

TABLE 4 Exemplary sequences of human CD137. SEQ ID DescriptionAmino Acid Sequence NO: CD137 signal MGNSCYNIVATLLLVLNFERTRS 23 peptideExemplary CD137 LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRT 24 extracellularCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGC domain sequenceSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSV TPPAPAREPGHSPQ ExemplaryMGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCD 25 immature CD137NNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRK full length sequenceECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFM RPVQTTQEEDGCSCRFPEEEEGGCELCD137 extracellular FNDQKRGICRPWTNCSL 26 epitope sequence #1CD137 extracellular FNDQKRGICRPWTNCSLDGKSVLVNGTKERD 27epitope sequence #2 CD137 extracellular TPGFHCLGAG 28epitope sequence #3 CD137 extracellular KQGQEL 29 epitope sequence #4CD137 extracellular LTKKGCKDCCFGTFNDQKRGICRPWTNC 30 epitope sequence #5CD137 extracellular FNDQKRGICRPWTNC 31 epitope sequence #6Exemplary mature LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRT 33CD137 full length CDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGC sequenceSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCEL CD137 fragmentDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCD 34 CD137 CRD4CCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDV 42 sequence VC CD137 fragment KRGI43

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), the antibody comprising a VH domain comprising one,two, or all three of the CDRs of a VH domain set forth in Table 1herein. In certain embodiments, the antibody comprises the CDRH1 of oneof VH domains set forth in Table 1. In certain embodiments, the antibodycomprises the CDRH2 of one of the VH domains set forth in Table 1. Incertain embodiments, the antibody comprises the CDRH3 of one of the VHdomains set forth in Table 1.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), the antibody comprising a VL domain comprising one,two, or all three of the CDRs of a VL domain disclosed in Table 1herein. In certain embodiments, the antibody comprises the CDRL1 of oneof VL domains set forth in Table 1. In certain embodiments, the antibodycomprises the CDRL2 of one of the VL domains set forth in Table 1. Incertain embodiments, the antibody comprises the CDRL3 of one of the VLdomains set forth in Table 1.

In certain embodiments, the CDRs of an antibody can be determinedaccording to Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) andKabat et al., Sequences of protein of immunological interest (1991),each of which is herein incorporated by reference in its entirety. Incertain embodiments, the light chain CDRs of an antibody are determinedaccording to Kabat and the heavy chain CDRs of an antibody aredetermined according to MacCallum (supra).

In certain embodiments, the CDRs of an antibody can be determinedaccording to the Chothia numbering scheme, which refers to the locationof immunoglobulin structural loops (see, e.g., Chothia C & Lesk A M,(1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817;Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Pat. No.7,709,226, all of which are herein incorporated by reference in theirentireties). Typically, when using the Kabat numbering convention, theChothia CDRH1 loop is present at heavy chain amino acids 26 to 32, 33,or 34, the Chothia CDRH2 loop is present at heavy chain amino acids 52to 56, and the Chothia CDRH3 loop is present at heavy chain amino acids95 to 102, while the Chothia CDRL1 loop is present at light chain aminoacids 24 to 34, the Chothia CDRL2 loop is present at light chain aminoacids 50 to 56, and the Chothia CDRL3 loop is present at light chainamino acids 89 to 97. The end of the Chothia CDRH1 loop when numberedusing the Kabat numbering convention varies between H32 and H34depending on the length of the loop (this is because the Kabat numberingscheme places the insertions at H35A and H35B; if neither 35A nor 35B ispresent, the loop ends at 32; if only 35A is present, the loop ends at33; if both 35A and 35B are present, the loop ends at 34).

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), the antibody comprising the Chothia VH CDRs of a VHdisclosed in Table 1 herein. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toCD137 (e.g., human CD137 or cynomolgus CD137), the antibody comprisingthe Chothia VL CDRs of a VL disclosed in Table 1 herein. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to CD137 (e.g., human CD137 or cynomolgus CD137), theantibody comprising the Chothia VH CDRs and Chothia VL CDRs of anantibody disclosed in Table 1 herein. In certain embodiments, antibodiesthat specifically bind to CD137 (e.g., human CD137 or cynomolgus CD137)comprise one or more CDRs, in which the Chothia and Kabat CDRs have thesame amino acid sequence. In certain embodiments, the instant disclosureprovides an isolated antibody that specifically binds to CD137 (e.g.,human CD137 or cynomolgus CD137) and comprises combinations of KabatCDRs and Chothia CDRs.

In certain embodiments, the CDRs of an antibody can be determinedaccording to MacCallum R M et al., (1996) J Mol Biol 262: 732-745,herein incorporated by reference in its entirety. See also, e.g., MartinA. “Protein Sequence and Structure Analysis of Antibody VariableDomains,” in Antibody Engineering, Kontermann and Dithel, eds., Chapter31, pp. 422-439, Springer-Verlag, Berlin (2001), herein incorporated byreference in its entirety.

In certain embodiments, the CDRs of an antibody can be determinedaccording to the IMGT numbering system as described in Lefranc M-P,(1999) The Immunologist 7: 132-136 and Lefranc M-P et al., (1999)Nucleic Acids Res 27: 209-212, each of which is herein incorporated byreference in its entirety. According to the IMGT numbering scheme, CDRH1is at positions 26 to 35, CDRH2 is at positions 51 to 57, CDRH3 is atpositions 93 to 102, CDRL1 is at positions 27 to 32, CDRL2 is atpositions 50 to 52, and CDRL3 is at positions 89 to 97.

In certain embodiments, the instant disclosure provides antibodies thatspecifically bind to CD137 (e.g., human CD137 or cynomolgus CD137) andcomprise CDRs of an antibody disclosed in Table 1 herein, as determinedby the IMGT numbering system, for example, as described in Lefranc M-P(1999) supra and Lefranc M-P et al., (1999) supra.

In certain embodiments, the CDRs of an antibody can be determinedaccording to the AbM numbering scheme, which refers to AbM hypervariableregions, which represent a compromise between the Kabat CDRs and Chothiastructural loops, and are used by Oxford Molecular's AbM antibodymodeling software (Oxford Molecular Group, Inc.), herein incorporated byreference in its entirety. In a particular embodiment, the instantdisclosure provides antibodies that specifically bind to CD137 (e.g.,human CD137 or cynomolgus CD137) and comprise CDRs of an antibodydisclosed in Table 1 herein as determined by the AbM numbering scheme.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), wherein the antibody comprises a heavy chain variableregion comprising the CDRH1, CDRH2, and CDRH3 region amino acidsequences of a VH, and a light chain variable region comprising theCDRL1, CDRL2, and CDRL3 region amino acid sequences of a VL, wherein theamino acid sequences of the VH and the VL are set forth in SEQ ID NOs: 7and 8; 63 and 8; 64 and 66; 7 and 67; or 65 and 68, respectively, andwherein each CDR is defined in accordance with the MacCallum definition,the Kabat definition, the Chothia definition, the combination of theKabat definition and the Chothia definition, the IMGT numbering system,or the AbM definition of CDR. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toCD137 (e.g., human CD137 or cynomolgus CD137), wherein the antibodycomprises a heavy chain variable region comprising the CDRH1, CDRH2, andCDRH3 region amino acid sequences of a VH domain set forth in SEQ ID NO:7, and a light chain variable region comprising the CDRL1, CDRL2, andCDRL3 region amino acid sequences of a VL domain set forth in SEQ ID NO:8, wherein each CDR is defined in accordance with the MacCallumdefinition, the Kabat definition, the Chothia definition, thecombination of the Kabat definition and the Chothia definition, the IMGTnumbering system, or the AbM definition of CDR.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to CD137 (e.g., human CD137 or cynomolgusCD137), the antibody comprising a heavy chain variable region (VH)comprising complementarity determining regions (CDRs) CDRH1, CDRH2, andCDRH3, and a light chain variable region (VL) comprising CDRs CDRL1,CDRL2, and CDRL3, wherein:

(SEQ ID NO: 82)(a) CDRH1 comprises the amino acid sequence of X₁X₂X₃X₄H,wherein

X₁ is G, A, D, E, L, N, Q, R, S, or W;

X₂ is Y, F, H, N, R, or S;

X₃ is Y or H; and

X₄ is M, I, T, or V;

(SEQ ID NO: 2)(b) CDRH2 comprises the amino acid sequence of WINPNSGGTNYAQKFQG;(SEQ ID NO: 83)(c) CDRH3 comprises the amino acid sequence of X₁PX₂YX₃GX₄GLX₅X₆,wherein

X₁ is E or G;

X₂ is G, A, R, or S;

X₃ is Y, F, H, or S;

X₄ is S, A, or T;

X₅ is D or G; and

X₆ is Y or H;

(SEQ ID NO: 4)(d) CDRL1 comprises the amino acid sequence of GGDDIGDKRVH;(SEQ ID NO: 5) (e) CDRL2 comprises the amino acid sequence of EDRYRPS;and/or (SEQ ID NO: 84)(f) CDRL3 comprises the amino acid sequence of QX₁WX₂X₃X₄X₅X₆X₇PGV,wherein

X₁ is V or I;

X₂ is D, A, E, G, H, N, or Y;

X₃ is S, A, E, F, L, P, R, T, W, or Y;

X₄ is S, A, L, M, or R;

X₅ is S, A, F, G, L, P, Q, R, or T;

X₆ is D, E, H, V, or Y; and

X₇ is H or Y.

In certain embodiments, CDRH1 comprises the amino acid sequence ofX₁X₂X₃X₄H (SEQ ID NO: 82), wherein X₁ is G, A, D, E, L, N, Q, R, S, orW; X₂ is Y, F, H, N, R, or S; X₃ is Y or H; and X₄ is M, I, T, or V. Incertain embodiments, CDRH3 comprises the amino acid sequence ofX₁PX₂YX₃GX₄GLX₅X₆ (SEQ ID NO: 83), wherein X₁ is E or G; X₂ is G, A, R,or S; X₃ is Y, F, H, or S; X₄ is S, A, or T; X₅ is D or G; and X₆ is Yor H. In certain embodiments, CDRL3 comprises the amino acid sequence ofQX₁WX₂X₃X₄X₅X₆X₇PGV (SEQ ID NO: 84), wherein X₁ is V or I; X₂ is D, A,E, G, H, N, or Y; X₃ is S, A, E, F, L, P, R, T, W, or Y; X₄ is S, A, L,M, or R; X₅ is S, A, F, G, L, P, Q, R, or T; X₆ is D, E, H, V, or Y; andX₇ is H or Y. In certain embodiments,

(a) CDRH1 comprises the amino acid sequence of (SEQ ID NO: 82)X₁X₂X₃X₄H,wherein

X₁ is G, A, D, E, L, N, Q, R, S, or W;

X₂ is Y, F, H, N, R, or S;

X₃ is Y or H; and

X₄ is M, I, T, or V;

(b) CDRH2 comprises the amino acid sequence of (SEQ ID NO: 2)WINPNSGGTNYAQKFQG; (c) CDRH3 comprises the amino acid sequence of(SEQ ID NO: 83) X₁PX₂YX₃GX₄GLX₅X₆,wherein

X₁ is E or G;

X₂ is G, A, R, or S;

X₃ is Y, F, H, or S;

X₄ is S, A, or T;

X₅ is D or G; and

X₆ is Y or H;

(d) CDRL1 comprises the amino acid sequence of (SEQ ID NO: 4)GGDDIGDKRVH; (e) CDRL2 comprises the amino acid sequence of(SEQ ID NO: 5) EDRYRPS; and(f) CDRL3 comprises the amino acid sequence of (SEQ ID NO: 84)QX₁WX₂X₃X₄X₅X₆X₇PGV,wherein

X₁ is V or I;

X₂ is D, A, E, G, H, N, or Y;

X₃ is S, A, E, F, L, P, R, T, W, or Y;

X₄ is S, A, L, M, or R;

X₅ is S, A, F, G, L, P, Q, R, or T;

X₆ is D, E, H, V, or Y; and

X₇ is H or Y.

In certain embodiments,

(a) CDRH1 comprises the amino acid sequence of (SEQ ID NO: 85) X₁X₂YX₃H,wherein

X₁ is G, A, D, L, R, S, or W;

X₂ is Y, F, H, or N; and

X₃ is M or V;

(b) CDRH3 comprises the amino acid sequence of (SEQ ID NO: 86)EPGYX₁GX₂GLDX₃,wherein

X₁ is Y or F;

X₂ is S or T; and

X₃ is Y or H; and/or

(c) CDRL3 comprises the amino acid sequence of (SEQ ID NO: 87)QVWX₁X₂X₃X₄X₅X₆PGV,wherein

X₁ is D, A, E, H, N, or Y;

X₂ is S, A, E, L, R, or T;

X₃ is S, A, L, or R;

X₄ is S, A, F, G, L, P, Q, or R;

X₅ is D, E, or V; and

X₆ is H or Y.

In certain embodiments, CDRH1 comprises the amino acid sequence ofX₁X₂YX₃H (SEQ ID NO: 85), wherein X₁ is G, A, D, L, R, S, or W; X₂ is Y,F, H, or N; and X₃ is M or V. In certain embodiments, CDRH3 comprisesthe amino acid sequence of EPGYX₁GX₂GLDX₃ (SEQ ID NO: 86), wherein X₁ isY or F; X₂ is S or T; and X₃ is Y or H. In certain embodiments, CDRL3comprises the amino acid sequence of QVWX₁X₂X₃X₄X₅X₆PGV (SEQ ID NO: 87),wherein X₁ is D, A, E, H, N, or Y; X₂ is S, A, E, L, R, or T; X₃ is S,A, L, or R; X₄ is S, A, F, G, L, P, Q, or R; X₅ is D, E, or V; and X₆ isH or Y. In certain embodiments,

(a) CDRH1 comprises the amino acid sequence of X₁X₂YX₃H (SEQ ID NO: 85),wherein

X₁ is G, A, D, L, R, S, or W;

X₂ is Y, F, H, or N; and

X₃ is M or V;

(b) CDRH3 comprises the amino acid sequence of (SEQ ID NO: 86)EPGYX₁GX₂GLDX₃,wherein

X₁ is Y or F;

X₂ is S or T; and

X₃ is Y or H; and

(c) CDRL3 comprises the amino acid sequence of (SEQ ID NO: 87)QVWX₁X₂X₃X₄X₅X₆PGV,wherein

X₁ is D, A, E, H, N, or Y;

X₂ is S, A, E, L, R, or T;

X₃ is S, A, L, or R;

X₄ is S, A, F, G, L, P, Q, or R;

X₅ is D, E, or V; and

X₆ is H or Y.

In certain embodiments, the CDRH1 comprises the amino acid sequence ofSEQ ID NO: 1. In certain embodiments, the CDRH3 comprises the amino acidsequence of SEQ ID NO: 3 or 59. In certain embodiments, the CDRL3 theamino acid sequence of SEQ ID NO: 6, 61, 62, or 63.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), wherein the antibody comprises a VH domain comprisingthe CDRH1, CDRH2 and CDRH3 amino acid sequences set forth in SEQ ID NOs:1, 2, and 3; or 1, 2, and 59, respectively. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD137 (e.g., human CD137 or cynomolgus CD137), wherein the antibodycomprises a VL domain comprising the CDRL1, CDRL2 and CDRL3 amino acidsequences set forth in SEQ ID NOs: 4, 5, and 6; 4, 5, and 60; 4, 5, and61; or 4, 5, and 62, respectively. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toCD137 (e.g., human CD137 or cynomolgus CD137), wherein the antibodycomprises a heavy chain variable region comprising CDRH1, CDRH2, andCDRH3 regions, and a light chain variable region comprising CDRL1,CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2,and CDRL3 regions comprise the amino acid sequences set forth in SEQ IDNOs: 1, 2, 3, 4, 5, and 6; 1, 2, 59, 4, 5, and 6; 1, 2, 3, 4, 5, and 60;1, 2, 3, 4, 5, and 61; or 1, 2, 3, 4, 5, and 62, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), the antibody comprising:

(SEQ ID NO: 1) (a) a CDRH1 comprises the amino acid sequence of GYYMH;(SEQ ID NO: 2) (b)a CDRH2 comprises the amino acid sequence of WINPNSGGTNYAQKFQG;(SEQ ID NO: 3) (c)a CDRH3 comprises the amino acid sequence of EPGYYGSGLDY; (SEQ ID NO: 4)(d) a CDRL1 comprises the amino acid sequence of GGDDIGDKRVH;(SEQ ID NO: 5) (e) a CDRL2 comprises the amino acid sequence of EDRYRPS;and/or (SEQ ID NO: 6) (f)a CDRL3 comprises the amino acid sequence of QVWDSSSDHPGV.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), wherein the antibody comprises a VH domain comprisingthe CDRH1, CDRH2 and CDRH3 amino acid sequences set forth in SEQ ID NOs:1, 2, and 3, respectively. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toCD137 (e.g., human CD137 or cynomolgus CD137), wherein the antibodycomprises a VL domain comprising the CDRL1, CDRL2 and CDRL3 amino acidsequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to CD137 (e.g., human CD137 or cynomolgus CD137),wherein the antibody comprises a heavy chain variable region comprisingCDRH1, CDRH2, and CDRH3 regions, and a light chain variable regioncomprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2,CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequencesset forth in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), comprising a heavy chain variable region comprisingan amino acid sequence of SEQ ID NO: 7. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD137 (e.g., human CD137 or cynomolgus CD137), comprising a heavychain variable region comprising an amino acid sequence that is at least75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequenceset forth in SEQ ID NO: 7. In certain embodiments, the heavy chainvariable region comprises the amino acid sequence of SEQ ID NO: 63, 64,or 65.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), comprising a light chain variable region comprisingan amino acid sequence of SEQ ID NO: 8. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD137 (e.g., human CD137 or cynomolgus CD137), comprising a lightchain variable region comprising an amino acid sequence that is at least75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequenceset forth in SEQ ID NO: 8. In certain embodiments, the light chainvariable region comprises the amino acid sequence of SEQ ID NO: 66, 67,or 68.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), comprising a heavy chain variable region comprisingan amino acid sequence of SEQ ID NO: 7, and a light chain variableregion comprising an amino acid sequence of SEQ ID NO: 8. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to CD137 (e.g., human CD137 or cynomolgus CD137),comprising a heavy chain variable region comprising an amino acidsequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., atleast 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%)identical to the amino acid sequence set forth in SEQ ID NO: 7, and alight chain variable region comprising an amino acid sequence that is atleast 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acidsequence set forth in SEQ ID NO: 8. In certain embodiments, the heavychain variable region and the light chain variable region comprise theamino acid sequences of SEQ ID NOs: 63 and 8; 64 and 66; 7 and 67; or 65and 68, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), comprising a heavy chain variable region having anamino acid sequence derived from a human IGHV1-2 germline sequence(e.g., IGHV1-2*02, e.g., having the amino acid sequence of SEQ ID NO:40). One or more regions selected from framework 1, framework 2,framework 3, CDRH1, and CDRH2 (e.g., two, three, four or five of theseregions) can be derived from a human IGHV1-2 germline sequence (e.g.,IGHV1-2*02, e.g., having the amino acid sequence of SEQ ID NO: 40). Inone embodiment, framework 1, framework 2, framework 3, CDRH1, and CDRH2are all derived from a human IGHV1-2 germline sequence (e.g.,IGHV1-2*02, e.g., having the amino acid sequence of SEQ ID NO: 40). Incertain embodiments, the heavy chain variable region comprises the aminoacid sequence set forth in SEQ ID NO: 3.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), comprising a light chain variable region having anamino acid sequence derived from a human IGLV3-21 germline sequence(e.g., IGLV3-21*02, e.g., having the amino acid sequence of SEQ ID NO:41, or IGLV3-21*03). One or more regions selected from framework 1,framework 2, framework 3, CDRL1, and CDRL2 (e.g., two, three, four orfive of these regions) can be derived from a human IGLV3-21 germlinesequence (e.g., IGLV3-21*02, e.g., having the amino acid sequence of SEQID NO: 41, or IGLV3-21*03). In one embodiment, framework 1, framework 2,framework 3, CDRL1, and CDRL2 are all derived from a human IGLV3-21germline sequence (e.g., IGLV3-21*02, e.g., having the amino acidsequence of SEQ ID NO: 41, or IGLV3-21*03). In certain embodiments, thelight chain variable region comprises the amino acid sequence set forthin SEQ ID NO: 6.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), comprising a heavy chain variable region having anamino acid sequence derived from a human IGHV1-2 germline sequence(e.g., IGHV1-2*02, e.g., having the amino acid sequence of SEQ ID NO:40), and a light chain variable region having an amino acid sequencederived from a human IGLV3-21 germline sequence (e.g., IGLV3-21*02,e.g., having the amino acid sequence of SEQ ID NO: 41, or IGLV3-21*03).In certain embodiments, the heavy chain variable region comprises theamino acid sequence set forth in SEQ ID NO: 3, and the light chainvariable region comprises the amino acid sequence set forth in SEQ IDNO: 6.

In certain embodiments, the instant disclosure provides an isolatedantibody that cross-competes for binding to CD137 (e.g., human CD137 orcynomolgus CD137) with an antibody comprising the heavy and light chainvariable region amino acid sequences set forth in SEQ ID NOs: 7 and 8,respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that binds to the same or an overlapping epitope of CD137(e.g., an epitope of human CD137 or an epitope of cynomolgus CD137) asan antibody described herein, e.g., an antibody comprising the heavy andlight chain variable region amino acid sequences set forth in SEQ IDNOs: 7 and 8, respectively. In certain embodiments, the epitope of anantibody can be determined by, e.g., NMR spectroscopy, surface plasmonresonance (BIAcore®), X-ray diffraction crystallography studies, ELISAassays, hydrogen/deuterium exchange coupled with mass spectrometry(e.g., liquid chromatography electrospray mass spectrometry),array-based oligo-peptide scanning assays, and/or mutagenesis mapping(e.g., site-directed mutagenesis mapping). For X-ray crystallography,crystallization may be accomplished using any of the known methods inthe art (e.g., Giegé R et al., (1994) Acta Crystallogr D BiolCrystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen N E (1997) Structure 5: 1269-1274; McPherson A (1976) JBiol Chem 251: 6300-6303, all of which are herein incorporated byreference in their entireties). Antibody:antigen crystals may be studiedusing well known X-ray diffraction techniques and may be refined usingcomputer software such as X-PLOR (Yale University, 1992, distributed byMolecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114& 115, eds Wyckoff H W et al.; U.S. Patent Application No.2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D BiolCrystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter C W; Roversi P et al., (2000) Acta Crystallogr D BiolCrystallogr 56(Pt 10): 1316-1323, all of which are herein incorporatedby reference in their entireties). Mutagenesis mapping studies may beaccomplished using any method known to one of skill in the art. See,e.g., Champe M et al., (1995) supra and Cunningham B C & Wells J A(1989) supra for a description of mutagenesis techniques, includingalanine scanning mutagenesis techniques. In a specific embodiment, theepitope of an antibody is determined using alanine scanning mutagenesisstudies. In addition, antibodies that recognize and bind to the same oroverlapping epitopes of CD137 (e.g., human CD137 or cynomolgus CD137)can be identified using routine techniques such as an immunoassay, forexample, by showing the ability of one antibody to block the binding ofanother antibody to a target antigen, i.e., a competitive binding assay.Competition binding assays also can be used to determine whether twoantibodies have similar binding specificity for an epitope. Competitivebinding can be determined in an assay in which the immunoglobulin undertest inhibits specific binding of a reference antibody to a commonantigen, such as CD137 (e.g., human CD137 or cynomolgus CD137). Numeroustypes of competitive binding assays are known, for example: solid phasedirect or indirect radioimmunoassay (MA), solid phase direct or indirectenzyme immunoassay (EIA), sandwich competition assay (see Stahli C etal., (1983) Methods Enzymol 9: 242-253); solid phase directbiotin-avidin EIA (see Kirkland T N et al., (1986) J Immunol 137:3614-9); solid phase direct labeled assay, solid phase direct labeledsandwich assay (see Harlow E & Lane D, (1988) Antibodies: A LaboratoryManual, Cold Spring Harbor Press); solid phase direct label RIA using1-125 label (see Morel G A et al., (1988) Mol Immunol 25(1): 7-15);solid phase direct biotin-avidin EIA (see Cheung R C et al., (1990)Virology 176: 546-52); and direct labeled RIA (see Moldenhauer G et al.,(1990) Scand J Immunol 32: 77-82), all of which are herein incorporatedby reference in their entireties. Typically, such an assay involves theuse of purified antigen (e.g., CD137, such as human CD137 or cynomolgusCD137) bound to a solid surface or cells bearing either of these, anunlabeled test immunoglobulin and a labeled reference immunoglobulin.Competitive inhibition can be measured by determining the amount oflabel bound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess.Usually, when a competing antibody is present in excess, it will inhibitspecific binding of a reference antibody to a common antigen by at least50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more. A competition bindingassay can be configured in a large number of different formats usingeither labeled antigen or labeled antibody. In a common version of thisassay, the antigen is immobilized on a 96-well plate. The ability ofunlabeled antibodies to block the binding of labeled antibodies to theantigen is then measured using radioactive or enzyme labels. For furtherdetails see, for example, Wagener C et al., (1983) J Immunol 130:2308-2315; Wagener C et al., (1984) J Immunol Methods 68: 269-274;Kuroki M et al., (1990) Cancer Res 50: 4872-4879; Kuroki M et al.,(1992) Immunol Invest 21: 523-538; Kuroki M et al., (1992) Hybridoma 11:391-407 and Antibodies: A Laboratory Manual, Ed Harlow E & Lane Deditors supra, pp. 386-389, all of which are herein incorporated byreference in their entireties.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human CD137, wherein (a)the antibody specifically binds to a protein comprising the amino acidsequence of SEQ ID NO: 37 and (b) the antibody does not specificallybind to a protein comprising the amino acid sequence of SEQ ID NO: 38.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human CD137, wherein theantibody specifically binds to a protein having the amino acid sequenceof SEQ ID NO: 38 with a lower affinity than to a protein having theamino acid sequence of SEQ ID NO: 37.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human CD137, wherein theantibody does not specifically bind to a protein having the amino acidsequence of SEQ ID NO: 38.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human CD137, wherein thebinding between the antibody and a protein having the amino acidsequence of SEQ ID NO: 38 is substantially weakened relative to thebinding between the antibody and a protein having the amino acidsequence of SEQ ID NO: 37.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human CD137, wherein theantibody exhibits, as compared to binding to a protein having the aminoacid sequence of SEQ ID NO: 37, reduced or absent binding to a proteinhaving the amino acid sequence of SEQ ID NO: 38.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to the same epitope of humanCD137 as any antibody of the present invention. In certain embodiments,the antibody specifically binds to a protein having the amino acidsequence of SEQ ID NO: 38 with a lower affinity than to a protein havingthe amino acid sequence of SEQ ID NO: 37. In certain embodiments, theantibody does not specifically bind to a protein having the amino acidsequence of SEQ ID NO: 38. In certain embodiments, the binding betweenthe antibody and a protein having the amino acid sequence of SEQ ID NO:38 is substantially weakened relative to the binding between theantibody and a protein having the amino acid sequence of SEQ ID NO: 37.In one embodiment, the antibody exhibits, as compared to binding to aprotein having the amino acid sequence of SEQ ID NO: 37, reduced orabsent binding to a protein having the amino acid sequence of SEQ ID NO:38.

In certain embodiments, the isolated antibody binds to an epitopelocated within a region of human CD137 comprising the amino acidsequence of any one of SEQ ID NOs: 26-31 and 43. In certain embodiments,the isolated antibody binds to an epitope located within a region ofhuman CD137 consisting essentially of the amino acid sequence of any oneof SEQ ID NOs: 26-31 and 43. In certain embodiments, the isolatedantibody binds to an epitope located within a region of human CD137consisting of the amino acid sequence of any one of SEQ ID NOs: 26-31and 43. In certain embodiments, the isolated antibody binds to adiscontinuous epitope located within a region of human CD137 comprisinga plurality of amino acid sequences, each of the plurality of amino acidsequences consisting of, consisting essentially of, or comprising theamino acid sequence of any one of SEQ ID NOs: 26-31 and 43.

In certain embodiments, the isolated antibody binds to an epitopelocated within a region of human CD137 comprising, consistingessentially of, or consisting of the amino acid sequence set forth inSEQ ID NO: 26. In another aspect, the instant disclosure provides anantibody that, when bound to a human CD137 protein or fragment thereof,reduces hydrogen/deuterium exchange in a region consisting of the aminoacid sequence set forth in SEQ ID NO: 26 relative to hydrogen/deuteriumexchange in the region consisting of the amino acid sequence set forthin SEQ ID NO: 26 in the absence of the antibody, as determined by ahydrogen/deuterium exchange assay. In certain embodiments, the reductionin hydrogen/deuterium exchange is measured using hydrogen-deuteriumexchange (HDX), for example as described herein in the examples.

In certain embodiments, the isolated antibody binds to an epitopelocated within a region of human CD137 comprising, consistingessentially of, or consisting of the amino acid sequence set forth inSEQ ID NO: 27. In another aspect, the instant disclosure provides anantibody that, when bound to a human CD137 protein or fragment thereof,reduces hydrogen/deuterium exchange in a region consisting of the aminoacid sequence set forth in SEQ ID NO: 27 relative to hydrogen/deuteriumexchange in the region consisting of the amino acid sequence set forthin SEQ ID NO: 27 in the absence of the antibody, as determined by ahydrogen/deuterium exchange assay. In certain embodiments, the reductionin hydrogen/deuterium exchange is measured using hydrogen-deuteriumexchange (HDX), for example as described herein in the examples.

In certain embodiments, the isolated antibody binds to an epitopelocated within a region of human CD137 comprising, consistingessentially of, or consisting of the amino acid sequence set forth inSEQ ID NO: 28. In another aspect, the instant disclosure provides anantibody that, when bound to a human CD137 protein or fragment thereof,reduces hydrogen/deuterium exchange in a region consisting of the aminoacid sequence set forth in SEQ ID NO: 28 relative to hydrogen/deuteriumexchange in the region consisting of the amino acid sequence set forthin SEQ ID NO: 28 in the absence of the antibody, as determined by ahydrogen/deuterium exchange assay. In certain embodiments, the reductionin hydrogen/deuterium exchange is measured using hydrogen-deuteriumexchange (HDX), for example as described herein in the examples.

In certain embodiments, the isolated antibody binds to an epitopelocated within a region of human CD137 comprising, consistingessentially of, or consisting of the amino acid sequence set forth inSEQ ID NO: 29. In another aspect, the instant disclosure provides anantibody that, when bound to a human CD137 protein or fragment thereof,reduces hydrogen/deuterium exchange in a region consisting of the aminoacid sequence set forth in SEQ ID NO: 29 relative to hydrogen/deuteriumexchange in the region consisting of the amino acid sequence set forthin SEQ ID NO: 29 in the absence of the antibody, as determined by ahydrogen/deuterium exchange assay. In certain embodiments, the reductionin hydrogen/deuterium exchange is measured using hydrogen-deuteriumexchange (HDX), for example as described herein in the examples.

In certain embodiments, the isolated antibody binds to an epitopelocated within a region of human CD137 comprising, consistingessentially of, or consisting of the amino acid sequence set forth inSEQ ID NO: 30. In another aspect, the instant disclosure provides anantibody that, when bound to a human CD137 protein or fragment thereof,reduces hydrogen/deuterium exchange in a region consisting of the aminoacid sequence set forth in SEQ ID NO: 30 relative to hydrogen/deuteriumexchange in the region consisting of the amino acid sequence set forthin SEQ ID NO: 30 in the absence of the antibody, as determined by ahydrogen/deuterium exchange assay. In certain embodiments, the reductionin hydrogen/deuterium exchange is measured using hydrogen-deuteriumexchange (HDX), for example as described herein in the examples.

In certain embodiments, the isolated antibody binds to an epitopelocated within a region of human CD137 comprising, consistingessentially of, or consisting of the amino acid sequence set forth inSEQ ID NO: 31. In another aspect, the instant disclosure provides anantibody that, when bound to a human CD137 protein or fragment thereof,reduces hydrogen/deuterium exchange in a region consisting of the aminoacid sequence set forth in SEQ ID NO: 31 relative to hydrogen/deuteriumexchange in the region consisting of the amino acid sequence set forthin SEQ ID NO: 31 in the absence of the antibody, as determined by ahydrogen/deuterium exchange assay. In certain embodiments, the reductionin hydrogen/deuterium exchange is measured using hydrogen-deuteriumexchange (HDX), for example as described herein in the examples.

In certain embodiments, the isolated antibody binds to an epitopelocated within a region of human CD137 comprising, consistingessentially of, or consisting of the amino acid sequence of KRGI (SEQ IDNO: 43). In certain embodiments, the antibody binds to at least one, atleast two, or at least three amino acid residues of KRGI. In certainembodiments, the antibody binds to all four amino acid residues of KRGI.In another aspect, the instant disclosure provides an antibody that,when bound to a human CD137 protein or fragment thereof, reduceshydrogen/deuterium exchange in a region consisting of the amino acidsequence set forth in SEQ ID NO: 43 relative to hydrogen/deuteriumexchange in the region consisting of the amino acid sequence set forthin SEQ ID NO: 43 in the absence of the antibody, as determined by ahydrogen/deuterium exchange assay. In certain embodiments, the reductionin hydrogen/deuterium exchange is measured using hydrogen-deuteriumexchange (HDX), for example as described herein in the examples. Inanother aspect, the instant disclosure provides an antibody thatspecifically binds to human CD137 and does not substantially bind tomurine CD137. In certain embodiments, the antibody specifically binds toa protein comprising the amino acid sequence of SEQ ID NO: 46, and/ordoes not substantially bind to a protein comprising the amino acidsequence of SEQ ID NO: 45. In certain embodiments, the antibodyspecifically binds to a protein consisting of or consisting essentiallyof the amino acid sequence of SEQ ID NO: 46, and/or does notsubstantially bind to a protein consisting of or consisting essentiallyof the amino acid sequence of SEQ ID NO: 45. In certain embodiments, theantibody specifically binds to a protein consisting of or consistingessentially of the amino acid sequence of SEQ ID NO: 46, and does notsubstantially bind to a protein consisting of or consisting essentiallyof the amino acid sequence of SEQ ID NO: 45.

In another aspect, the instant disclosure provides an antibody orisolated antibody that binds, e.g., specifically binds, to the sameepitope of human CD137 as an antibody of the present invention. Incertain embodiments, the epitope is determined by hydrogen-deuteriumexchange (HDX), for example as described in the examples, or by proteinmutagenesis, for example as described in the examples.

In certain embodiments, the antibody comprises a VH and a VL, wherein ifthe antibody is formatted as a F(ab′)₂ comprising two of each of the VHand the VL, the F(ab′)₂ binds to an epitope located within a region ofhuman CD137 consisting of the amino acid sequence of SEQ ID NO: 27. Incertain embodiments, the antibody comprises a VH and a VL, wherein ifthe antibody is formatted as a F(ab′)₂ comprising two of each of the VHand the VL, the F(ab′)₂ substantially reduces (e.g., by at least 50%,60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) the exchange ofhydrogen with deuterium in a region of CD137 consisting of the aminoacid sequence of SEQ ID NO: 27 relative to the exchange of hydrogen withdeuterium in the same region in the absence of the F(ab′)₂, as measuredby a hydrogen/deuterium exchange assay.

In certain embodiments, the antibody comprises a VH and a VL, wherein ifthe antibody is formatted as a Fab comprising the VH and the VL, the Fabbinds to an epitope located within a region of human CD137 consisting ofthe amino acid sequence of SEQ ID NO: 26, and, optionally, an epitopelocated within a region of human CD137 consisting of the amino acidsequence of SEQ ID NOs: 28 and/or 29. In certain embodiments, theantibody comprises a VH and a VL, wherein if the antibody is formattedas a Fab comprising the VH and the VL, the Fab substantially reduces(e.g., by at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99%) the exchange of hydrogen with deuterium in a region of CD137consisting of the amino acid sequence of SEQ ID NO: 26 relative to theexchange of hydrogen with deuterium in the same region in the absence ofthe Fab, and, optionally, substantially reduces (e.g., by at least 50%,60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) the exchange ofhydrogen with deuterium in a region of CD137 consisting of the aminoacid sequence of SEQ ID NO: 28 and/or SEQ ID NO: 29 relative to theexchange of hydrogen with deuterium in the same regions in the absenceof the Fab, as measured by a hydrogen/deuterium exchange assay.

In certain embodiments, the antibody comprises a VH and a VL, wherein ifthe antibody is formatted as a F(ab′)₂ comprising two of chains, eachchain comprising the VH and the VL, the F(ab′)₂ substantially reduces(e.g., by at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99%) the exchange of hydrogen with deuterium in a region of CD137consisting of the amino acid sequence of SEQ ID NO: 34 relative to theexchange of hydrogen with deuterium in the same region in the absence ofthe F(ab′)₂, as measured by a hydrogen/deuterium exchange assay. Incertain embodiments, the antibody comprises a VH and a VL, wherein ifthe antibody is formatted as a Fab comprising the VH and the VL, the Fabdoes not substantially reduce (e.g., no reduction by more than 1%, 2%,3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30%) the exchange of hydrogen withdeuterium in a region of CD137 consisting of the amino acid sequence ofSEQ ID NO: 34 relative to the exchange of hydrogen with deuterium in thesame region in the absence of the Fab, as measured by ahydrogen/deuterium exchange assay.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137) and does not inhibit human CD137 from binding to humanCD137L. In certain embodiments, the binding of human CD137 to humanCD137L is not reduced by more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%,25%, or 30% in the presence of the antibody relative to the binding ofhuman CD137 to human CD137L in the absence of the antibody. In certainembodiments, the antibody does not inhibit a soluble fragment of humanCD137 from binding to a soluble fragment of human CD137L. In certainembodiments, the binding of a soluble fragment of human CD137 to asoluble fragment of human CD137L is not reduced by more than 1%, 2%, 3%,4%, 5%, 10%, 15%, 20%, 25%, or 30% in the presence of the antibodyrelative to the binding of a soluble fragment of human CD137 to asoluble fragment of human CD137L in the absence of the antibody. Incertain embodiments, the antibody does not inhibit a CD137-expressingcell from binding to a soluble fragment of human CD137L. In certainembodiments, the binding of a CD137-expressing cell to a solublefragment of human CD137L is not reduced by more than 1%, 2%, 3%, 4%, 5%,10%, 15%, 20%, 25%, or 30% in the presence of the antibody relative tothe binding of a CD137-expressing cell to a soluble fragment of humanCD137L in the absence of the antibody. In certain embodiments, theantibody does not inhibit a CD137-expressing cell from binding to aCD137L-expressing cell. In certain embodiments, the binding of aCD137-expressing cell to a CD137L-expressing cell is not reduced by morethan 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% in the presence ofthe antibody relative to the binding of a CD137-expressing cell to aCD137L-expressing cell in the absence of the antibody.

In certain embodiments, the antibody disclosed herein increases thelevel of CD137 multimerization (e.g., dimerization or trimerization)relative to the level of CD137 multimerization in the absence of theantibody by at least about 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, or more. Incertain embodiments, the multimeric CD137 is present in a complexcomprising CD137 and CD137L molecules (e.g., a complex comprising threeCD137L molecules and two CD137 molecules, or a complex comprising threeCD137L molecules and three CD137 molecules). In certain embodiments, thelevel of CD137 multimerization (e.g., dimerization or trimerization) ismeasured in an in vitro system comprising CD137 and CD137L molecules inequilibrium, optionally wherein the CD137 molecules are in a lipidbilayer membrane. In certain embodiments, the level of CD137multimerization (e.g., dimerization or trimerization) is measured in acell or on the plasma membrane of a cell. In certain embodiments, thelevel of CD137 multimerization (e.g., dimerization or trimerization) ismeasured using a soluble fragment of CD137.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), the antibody comprising a heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 9, 10, 11, 12, 13, 14, 49,50, 51, 52, 53, 54, 73, 74, 75, 76, 77, or 78. In certain embodiments,the antibody comprises a heavy chain comprising the amino acid sequenceset forth in SEQ ID NO: 9. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 10. In certain embodiments, the antibody comprises a heavychain comprising the amino acid sequence set forth in SEQ ID NO: 11. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 12. In certain embodiments,the antibody comprises a heavy chain comprising the amino acid sequenceset forth in SEQ ID NO: 13. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 14. In certain embodiments, the antibody comprises a heavychain comprising the amino acid sequence set forth in SEQ ID NO: 49. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 50. In certain embodiments,the antibody comprises a heavy chain comprising the amino acid sequenceset forth in SEQ ID NO: 51. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 52. In certain embodiments, the antibody comprises a heavychain comprising the amino acid sequence set forth in SEQ ID NO: 53. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 54. In certain embodiments,the antibody comprises a heavy chain comprising the amino acid sequenceset forth in SEQ ID NO: 73. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 74. In certain embodiments, the antibody comprises a heavychain comprising the amino acid sequence set forth in SEQ ID NO: 75. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 76. In certain embodiments,the antibody comprises a heavy chain comprising the amino acid sequenceset forth in SEQ ID NO: 77. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 78.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), the antibody comprising a light chain comprising theamino acid sequence set forth in SEQ ID NO: 21, 79, 80, or 81. Incertain embodiments, the antibody comprises a light chain comprising theamino acid sequence set forth in SEQ ID NO: 21. In certain embodiments,the antibody comprises a light chain comprising the amino acid sequenceset forth in SEQ ID NO: 79. In certain embodiments, the antibodycomprises a light chain comprising the amino acid sequence set forth inSEQ ID NO: 80. In certain embodiments, the antibody comprises a lightchain comprising the amino acid sequence set forth in SEQ ID NO: 81.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), the antibody comprising a heavy chain comprising theamino acid sequence of SEQ ID NO: 9, 10, 11, 12, 13, 14, 49, 50, 51, 52,53, 54, 73, 74, 75, 76, 77, or 78; and a light chain comprising theamino acid sequence of SEQ ID NO: 21, 79, 80, or 81. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to CD137 (e.g., human CD137 or cynomolgus CD137), theantibody comprising a heavy chain comprising the amino acid sequence ofSEQ ID NO: 9; and a light chain comprising the amino acid sequence ofSEQ ID NO: 21. In certain embodiments, the instant disclosure providesan isolated antibody that specifically binds to CD137 (e.g., human CD137or cynomolgus CD137), the antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 10; and a light chain comprisingthe amino acid sequence of SEQ ID NO: 21. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD137 (e.g., human CD137 or cynomolgus CD137), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 11; and a light chain comprising the amino acid sequence of SEQ IDNO: 21. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), the antibody comprising a heavy chain comprising theamino acid sequence of SEQ ID NO: 12; and a light chain comprising theamino acid sequence of SEQ ID NO: 21. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD137 (e.g., human CD137 or cynomolgus CD137), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 13; and a light chain comprising the amino acid sequence of SEQ IDNO: 21. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), the antibody comprising a heavy chain comprising theamino acid sequence of SEQ ID NO: 14; and a light chain comprising theamino acid sequence of SEQ ID NO: 21. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD137 (e.g., human CD137 or cynomolgus CD137), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 49; and a light chain comprising the amino acid sequence of SEQ IDNO: 21. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), the antibody comprising a heavy chain comprising theamino acid sequence of SEQ ID NO: 50; and a light chain comprising theamino acid sequence of SEQ ID NO: 21. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD137 (e.g., human CD137 or cynomolgus CD137), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 51; and a light chain comprising the amino acid sequence of SEQ IDNO: 21. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), the antibody comprising a heavy chain comprising theamino acid sequence of SEQ ID NO: 52; and a light chain comprising theamino acid sequence of SEQ ID NO: 21. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD137 (e.g., human CD137 or cynomolgus CD137), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 53; and a light chain comprising the amino acid sequence of SEQ IDNO: 21. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), the antibody comprising a heavy chain comprising theamino acid sequence of SEQ ID NO: 54; and a light chain comprising theamino acid sequence of SEQ ID NO: 21. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD137 (e.g., human CD137 or cynomolgus CD137), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 73; and a light chain comprising the amino acid sequence of SEQ IDNO: 21. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), the antibody comprising a heavy chain comprising theamino acid sequence of SEQ ID NO: 74; and a light chain comprising theamino acid sequence of SEQ ID NO: 21. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD137 (e.g., human CD137 or cynomolgus CD137), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 75; and a light chain comprising the amino acid sequence of SEQ IDNO: 79. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), the antibody comprising a heavy chain comprising theamino acid sequence of SEQ ID NO: 76; and a light chain comprising theamino acid sequence of SEQ ID NO: 79. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD137 (e.g., human CD137 or cynomolgus CD137), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 9; and a light chain comprising the amino acid sequence of SEQ IDNO: 80. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), the antibody comprising a heavy chain comprising theamino acid sequence of SEQ ID NO: 49; and a light chain comprising theamino acid sequence of SEQ ID NO: 80. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD137 (e.g., human CD137 or cynomolgus CD137), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 77; and a light chain comprising the amino acid sequence of SEQ IDNO: 81. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), the antibody comprising a heavy chain comprising theamino acid sequence of SEQ ID NO: 78; and a light chain comprising theamino acid sequence of SEQ ID NO: 81.

In certain embodiments, the antibody comprises a heavy chain consistingof the amino acid sequence of SEQ ID NO: 9, 10, 11, 12, 13, 14, 49, 50,51, 52, 53, 54, 73, 74, 75, 76, 77, or 78; and a light chain consistingof the amino acid sequence of SEQ ID NO: 21, 79, 80, or 81. In certainembodiments, the antibody comprises a heavy chain consisting of theamino acid sequence of SEQ ID NO: 9; and a light chain consisting of theamino acid sequence of SEQ ID NO: 21. In certain embodiments, theantibody comprises a heavy chain consisting of the amino acid sequenceof SEQ ID NO: 10; and a light chain consisting of the amino acidsequence of SEQ ID NO: 21. In certain embodiments, the antibodycomprises a heavy chain consisting of the amino acid sequence of SEQ IDNO: 11; and a light chain consisting of the amino acid sequence of SEQID NO: 21. In certain embodiments, the antibody comprises a heavy chainconsisting of the amino acid sequence of SEQ ID NO: 12; and a lightchain consisting of the amino acid sequence of SEQ ID NO: 21. In certainembodiments, the antibody comprises a heavy chain consisting of theamino acid sequence of SEQ ID NO: 13; and a light chain consisting ofthe amino acid sequence of SEQ ID NO: 21. In certain embodiments, theantibody comprises a heavy chain consisting of the amino acid sequenceof SEQ ID NO: 14; and a light chain consisting of the amino acidsequence of SEQ ID NO: 21. In certain embodiments, the antibodycomprises a heavy chain consisting of the amino acid sequence of SEQ IDNO: 49; and a light chain consisting of the amino acid sequence of SEQID NO: 21. In certain embodiments, the antibody comprises a heavy chainconsisting of the amino acid sequence of SEQ ID NO: 50; and a lightchain consisting of the amino acid sequence of SEQ ID NO: 21. In certainembodiments, the antibody comprises a heavy chain consisting of theamino acid sequence of SEQ ID NO: 51; and a light chain consisting ofthe amino acid sequence of SEQ ID NO: 21. In certain embodiments, theantibody comprises a heavy chain consisting of the amino acid sequenceof SEQ ID NO: 52; and a light chain consisting of the amino acidsequence of SEQ ID NO: 21. In certain embodiments, the antibodycomprises a heavy chain consisting of the amino acid sequence of SEQ IDNO: 53; and a light chain consisting of the amino acid sequence of SEQID NO: 21. In certain embodiments, the antibody comprises a heavy chainconsisting of the amino acid sequence of SEQ ID NO: 54; and a lightchain consisting of the amino acid sequence of SEQ ID NO: 21. In certainembodiments, the antibody comprises a heavy chain consisting of theamino acid sequence of SEQ ID NO: 73; and a light chain consisting ofthe amino acid sequence of SEQ ID NO: 21. In certain embodiments, theantibody comprises a heavy chain consisting of the amino acid sequenceof SEQ ID NO: 74; and a light chain consisting of the amino acidsequence of SEQ ID NO: 21. In certain embodiments, the antibodycomprises a heavy chain consisting of the amino acid sequence of SEQ IDNO: 75; and a light chain consisting of the amino acid sequence of SEQID NO: 79. In certain embodiments, the antibody comprises a heavy chainconsisting of the amino acid sequence of SEQ ID NO: 76; and a lightchain consisting of the amino acid sequence of SEQ ID NO: 79. In certainembodiments, the antibody comprises a heavy chain consisting of theamino acid sequence of SEQ ID NO: 9; and a light chain consisting of theamino acid sequence of SEQ ID NO: 80. In certain embodiments, theantibody comprises a heavy chain consisting of the amino acid sequenceof SEQ ID NO: 49; and a light chain consisting of the amino acidsequence of SEQ ID NO: 80. In certain embodiments, the antibodycomprises a heavy chain consisting of the amino acid sequence of SEQ IDNO: 77; and a light chain consisting of the amino acid sequence of SEQID NO: 81. In certain embodiments, the antibody comprises a heavy chainconsisting of the amino acid sequence of SEQ ID NO: 78; and a lightchain consisting of the amino acid sequence of SEQ ID NO: 81.

Any antibody format can be used in the antibodies disclosed herein. Incertain embodiments, the antibody is a single chain antibody orsingle-chain Fv (scFv). In certain embodiments, the antibody is a scFvfused with an Fc region (scFv-Fc). In certain embodiments, the antibodyis a Fab fragment. In certain embodiments, the antibody is a F(ab′)₂fragment.

In certain embodiments, the antibody disclosed herein is a multispecificantibody (e.g., a bispecific antibody) which specifically binds to CD137(e.g., human CD137 or cynomolgus CD137) and a second antigen.

In certain embodiments, the antibody disclosed herein is conjugated to asecond antibody that specifically binds to a second antigen. In certainembodiments, the antibody disclosed herein is covalently conjugated to asecond antibody. In certain embodiments, the antibody disclosed hereinis non-covalently conjugated to a second antibody. In certainembodiments, the antibody disclosed herein is cross-linked to a secondantibody. In certain embodiments, the second antigen is atumor-associated antigen (e.g., a polypeptide overexpressed in a tumor,a polypeptide derived from an oncovirus, a polypeptide comprising apost-translational modification specific to a tumor, a polypeptidespecifically mutated in a tumor). In certain embodiments, thetumor-associated antigen is EGFR (e.g., human EGFR), Her2 (e.g., humanHer2), or CD20 (e.g., human CD20).

In certain embodiments, the antibody disclosed herein is conjugated to acytotoxic agent, cytostatic agent, toxin, radionuclide, or detectablelabel. In certain embodiments, the cytotoxic agent is able to inducedeath or destruction of a cell in contact therewith. In certainembodiments, the cytostatic agent is able to prevent or substantiallyreduce proliferation and/or inhibits the activity or function of a cellin contact therewith. In certain embodiments, the cytotoxic agent orcytostatic agent is a chemotherapeutic agent. In certain embodiments,the radionuclide is selected from the group consisting of the isotopes³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶⁷Cu, ⁹⁰Y, 99Tc, ¹¹¹In,¹¹⁷Lu, ¹²¹I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁹⁸Au, ²¹¹At, ²¹³Bi, ²²⁵Ac and ¹⁸⁶Re. Incertain embodiments, the detectable label comprises a fluorescent moietyor a click chemistry handle.

Any immunoglobulin (Ig) constant region can be used in the antibodiesdisclosed herein. In certain embodiments, the Ig region is a human IgG,IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (e.g.,IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂), or any subclass (e.g., IgG_(2a)and IgG_(2b)) of immunoglobulin molecule.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), the antibody comprising a heavy chain constant regioncomprising the amino acid sequence of SEQ ID NO: 15, 16, 17, 18, 19, or20. In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137), the antibody comprising a light chain constant regioncomprising the amino acid sequence of SEQ ID NO: 22.

In certain embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of an antibodydescribed herein (e.g., CH2 domain (residues 231-340 of human IgG₁)and/or CH3 domain (residues 341-447 of human IgG₁) and/or the hingeregion, numbered according to the EU numbering system, to alter one ormore functional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding and/or antigen-dependentcellular cytotoxicity.

In certain embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the hinge region of the Fc region(CH1 domain) such that the number of cysteine residues in the hingeregion are altered (e.g., increased or decreased) as described in, e.g.,U.S. Pat. No. 5,677,425, herein incorporated by reference in itsentirety. The number of cysteine residues in the hinge region of the CH1domain may be altered to, e.g., facilitate assembly of the light andheavy chains, or to alter (e.g., increase or decrease) the stability ofthe antibody.

In a specific embodiment, one, two, or more amino acid mutations (e.g.,substitutions, insertions or deletions) are introduced into an IgGconstant domain, or FcRn-binding fragment thereof (preferably an Fc orhinge-Fc domain fragment) to alter (e.g., decrease or increase)half-life of the antibody in vivo. See, e.g., International PublicationNos. WO 02/060919; WO 98/23289; and WO 97/34631; and U.S. Pat. Nos.5,869,046, 6,121,022, 6,277,375 and 6,165,745, all of which are hereinincorporated by reference in their entireties, for examples of mutationsthat will alter (e.g., decrease or increase) the half-life of anantibody in vivo. In certain embodiments, one, two or more amino acidmutations (e.g., substitutions, insertions, or deletions) are introducedinto an IgG constant domain, or FcRn-binding fragment thereof(preferably an Fc or hinge-Fc domain fragment) to decrease the half-lifeof the antibody in vivo. In other embodiments, one, two or more aminoacid mutations (e.g., substitutions, insertions or deletions) areintroduced into an IgG constant domain, or FcRn-binding fragment thereof(preferably an Fc or hinge-Fc domain fragment) to increase the half-lifeof the antibody in vivo. In a specific embodiment, the antibodies mayhave one or more amino acid mutations (e.g., substitutions) in thesecond constant (CH2) domain (residues 231-340 of human IgG₁) and/or thethird constant (CH3) domain (residues 341-447 of human IgG₁), numberedaccording to the EU numbering system. In a specific embodiment, theconstant region of the IgG₁ of an antibody described herein comprises amethionine (M) to tyrosine (Y) substitution in position 252, a serine(S) to threonine (T) substitution in position 254, and a threonine (T)to glutamic acid (E) substitution in position 256, numbered according tothe EU numbering system. See U.S. Pat. No. 7,658,921, which is hereinincorporated by reference in its entirety. This type of mutant IgG,referred to as “YTE mutant” has been shown to display fourfold increasedhalf-life as compared to wild-type versions of the same antibody (seeDall'Acqua W F et al., (2006) J Biol Chem 281: 23514-24, which is hereinincorporated by reference in its entirety). In certain embodiments, anantibody comprises an IgG constant domain comprising one, two, three ormore amino acid substitutions of amino acid residues at positions251-257, 285-290, 308-314, 385-389, and 428-436, numbered according tothe EU numbering system.

In certain embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of an antibodydescribed herein (e.g., CH2 domain (residues 231-340 of human IgG₁)and/or CH3 domain (residues 341-447 of human IgG₁) and/or the hingeregion, numbered according to the EU numbering system, to increase ordecrease the affinity of the antibody for an Fc receptor (e.g., anactivated Fc receptor) on the surface of an effector cell. Mutations inthe Fc region of an antibody that decrease or increase the affinity ofan antibody for an Fc receptor and techniques for introducing suchmutations into the Fc receptor or fragment thereof are known to one ofskill in the art. Examples of mutations in the Fc receptor of anantibody that can be made to alter the affinity of the antibody for anFc receptor are described in, e.g., Smith P et al., (2012) PNAS 109:6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos.WO 02/060919; WO 98/23289; and WO 97/34631, all of which are hereinincorporated by reference in their entireties.

In certain embodiments, the antibody comprises a heavy chain constantregion that is a variant of a wild type heavy chain constant region,wherein the variant heavy chain constant region binds to FcγRIIB withhigher affinity than the wild type heavy chain constant region binds toFcγRIIB In certain embodiments, the variant heavy chain constant regionis a variant human heavy chain constant region, e.g., a variant humanIgG1, a variant human IgG2, or a variant human IgG4 heavy chain constantregion. In certain embodiments, the variant human IgG heavy chainconstant region comprises one or more of the following amino acidmutations, according to the EU numbering system: G236D, P238D, S239D,S267E, L328F, and L328E. In certain embodiments, the variant human IgGheavy chain constant region comprises a set of amino acid mutationsselected from the group consisting of: S267E and L328F; P238D and L328E;P238D and one or more substitutions selected from the group consistingof E233D, G237D, H268D, P271G, and A330R; P238D, E233D, G237D, H268D,P271G, and A330R; G236D and S267E; S239D and S267E; V262E, S267E, andL328F; and V264E, S267E, and L328F, according to the EU numberingsystem. In certain embodiments, the FcγRIIB is expressed on a cellselected from the group consisting of macrophages, monocytes, B cells,dendritic cells, endothelial cells, and activated T cells.

In a further embodiment, one, two, or more amino acid substitutions areintroduced into an IgG constant domain Fc region to alter the effectorfunction(s) of the antibody. For example, one or more amino acidsselected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and322, numbered according to the EU numbering system, can be replaced witha different amino acid residue such that the antibody has an alteredaffinity for an effector ligand but retains the antigen-binding abilityof the parent antibody. The effector ligand to which affinity is alteredcan be, for example, an Fc receptor or the Cl component of complement.This approach is described in further detail in U.S. Pat. Nos. 5,624,821and 5,648,260, each of which is herein incorporated by reference in itsentirety. In certain embodiments, the deletion or inactivation (throughpoint mutations or other means) of a constant region domain may reduceFc receptor binding of the circulating antibody thereby increasing tumorlocalization. See, e.g., U.S. Pat. Nos. 5,585,097 and 8,591,886, each ofwhich is herein incorporated by reference in its entirety, for adescription of mutations that delete or inactivate the constant domainand thereby increase tumor localization. In certain embodiments, one ormore amino acid substitutions may be introduced into the Fc region of anantibody described herein to remove potential glycosylation sites on theFc region, which may reduce Fc receptor binding (see, e.g., Shields R Let al., (2001) J Biol Chem 276: 6591-604, which is herein incorporatedby reference in its entirety). In various embodiments, one or more ofthe following mutations in the constant region of an antibody describedherein may be made: an N297A substitution; an N297Q substitution; aL235A substitution and a L237A substitution; a L234A substitution and aL235A substitution; a E233P substitution; a L234V substitution; a L235Asubstitution; a C236 deletion; a P238A substitution; a D265Asubstitution; a S267E substitution and L328F substitution; a A327Qsubstitution; or a P329A substitution, numbered according to the EUnumbering system. In certain embodiments, a mutation selected from thegroup consisting of D265A, P329A, and a combination thereof, numberedaccording to the EU numbering system, may be made in the constant regionof an antibody described herein.

In a specific embodiment, an antibody described herein comprises theconstant domain of an IgG₁ with an N297Q or N297A amino acidsubstitution, numbered according to the EU numbering system. In oneembodiment, an antibody described herein comprises the constant domainof an IgG₁ with a mutation selected from the group consisting of D265A,P329A, and a combination thereof, numbered according to the EU numberingsystem. In another embodiment, an antibody described herein comprisesthe constant domain of an IgG₁ with a mutation selected from the groupconsisting of L234A, L235A, and a combination thereof, numberedaccording to the EU numbering system. In certain embodiments, amino acidresidues in the constant region of an antibody described herein in thepositions corresponding to positions L234, L235, and D265 in a humanIgG₁ heavy chain, numbered according to the EU numbering system, are notL, L, and D, respectively. This approach is described in detail inInternational Publication No. WO 14/108483, which is herein incorporatedby reference in its entirety. In a particular embodiment, the aminoacids corresponding to positions L234, L235, and D265 in a human IgG₁heavy chain are F, E, and A; or A, A, and A, respectively, numberedaccording to the EU numbering system.

In certain embodiments, one or more amino acids selected from amino acidresidues 329, 331, and 322 in the constant region of an antibodydescribed herein, numbered according to the EU numbering system, can bereplaced with a different amino acid residue such that the antibody hasaltered C1q binding and/or reduced or abolished complement dependentcytotoxicity (CDC). This approach is described in further detail in U.S.Pat. No. 6,194,551 (Idusogie et al.), which is herein incorporated byreference in its entirety. In certain embodiments, one or more aminoacid residues within amino acid positions 231 to 238 in the N-terminalregion of the CH2 domain of an antibody described herein are altered tothereby alter the ability of the antibody to fix complement, numberedaccording to the EU numbering system. This approach is described furtherin International Publication No. WO 94/29351, which is hereinincorporated by reference in its entirety. In certain embodiments, theFc region of an antibody described herein is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by mutating one or more amino acids (e.g., introducingamino acid substitutions) at the following positions: 238, 239, 248,249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278,280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303,305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331,333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398,414, 416, 419, 430, 434, 435, 437, 438, or 439, numbered according tothe EU numbering system. This approach is described further inInternational Publication No. WO 00/42072, which is herein incorporatedby reference in its entirety.

In certain embodiments, an antibody described herein comprises theconstant region of an IgG4 antibody and the serine at amino acid residue228 of the heavy chain, numbered according to the EU numbering system,is substituted for proline. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toCD137 (e.g., human CD137 or cynomolgus CD137), the antibody comprising aheavy chain constant region comprising the amino acid sequence of SEQ IDNO: 20.

In certain embodiments, any of the constant region mutations ormodifications described herein can be introduced into one or both heavychain constant regions of an antibody described herein having two heavychain constant regions.

In certain embodiments of any of the aspects disclosed herein recitingSEQ ID NO: 7, 9, 10, 11, 12, 13, 14, 49, 50, 51, 52, 53, 54, 63, 64, 65,69, 70, 71, 72, 73, 74, 75, 76, 77, or 78, X in SEQ ID NO: 7, 9, 10, 11,12, 13, 14, 49, 50, 51, 52, 53, 54, 63, 64, 65, 69, 70, 71, 72, 73, 74,75, 76, 77, or 78 is glutamine (Q). In certain embodiments of any of theaspects disclosed herein reciting SEQ ID NO: 7, 9, 10, 11, 12, 13, 14,49, 50, 51, 52, 53, 54, 63, 64, 65, 69, 70, 71, 72, 73, 74, 75, 76, 77,or 78, X in SEQ ID NO: 7, 9, 10, 11, 12, 13, 14, 49, 50, 51, 52, 53, 54,63, 64, 65, 69, 70, 71, 72, 73, 74, 75, 76, 77, or 78 is pyroglutamate(pE).

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137) and functions as an agonist.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137) and increases or promotes CD137 (e.g., human CD137 orcynomolgus CD137) activity by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%,as assessed by methods described herein and/or known to one of skill inthe art, relative to CD137 (e.g., human CD137 or cynomolgus CD137)activity without any antibody or with an unrelated antibody (e.g., anantibody that does not specifically bind to CD137 (e.g., human CD137 orcynomolgus CD137)). In certain embodiments, the instant disclosureprovides an isolated antibody that specifically binds to CD137 (e.g.,human CD137 or cynomolgus CD137) and increases or promotes CD137 (e.g.,human CD137 or cynomolgus CD137) activity by at least about 1.2 fold,1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90fold, 100 fold, or more, as assessed by methods described herein and/orknown to one of skill in the art, relative to CD137 (e.g., human CD137or cynomolgus CD137) activity without any antibody or with an unrelatedantibody (e.g., an antibody that does not specifically bind to CD137(e.g., human CD137)). Non-limiting examples of CD137 (e.g., human CD137or cynomolgus CD137) activity can include CD137 (e.g., human CD137 orcynomolgus CD137) signaling, CD137 (e.g., human CD137 or cynomolgusCD137) binding to CD137 (e.g., human CD137 or cynomolgus CD137) ligand(e.g., CD137L (e.g., human CD137 or cynomolgus CD137) or a fragmentand/or fusion protein thereof), activation of a T cell (e.g., a T cellexpressing human CD137), increase of cytokine production (e.g., IL-2,IFN-γ and/or TNF-α), activation of a natural killer (NK) cell, increaseof CD137L activity, and activation of an antigen-presenting cell (APC)expressing CD137L. In specific embodiments, an increase in a CD137(e.g., human CD137 or cynomolgus CD137) activity is assessed asdescribed in the Examples, infra. In certain embodiments, the antibodyincreases or promotes an activity of CD137 (e.g., human CD137 orcynomolgus CD137) in the presence of a ligand of CD137 (e.g., CD137L(e.g., human CD137 or cynomolgus CD137) or a fragment and/or fusionprotein thereof).

In certain embodiments, the ability of the antibody to activate,increase, or promote an activity of CD137 (e.g., human CD137 orcynomolgus CD137) depends on the presence of crosslinking of theantibody. In certain embodiments, the antibody minimally increases orpromotes an activity of CD137 (e.g., human CD137 or cynomolgus CD137) inthe absence of crosslinking of the antibody. In certain embodiments, theantibody does not substantially increase or promote an activity of CD137(e.g., human CD137 or cynomolgus CD137) in the absence of crosslinkingof the antibody. In one embodiment, the antibody minimally induces NF-κBsignaling in a NF-κB reporter cell line, e.g., as measured in theexamples described herein, in the absence of crosslinking of theantibody. In one embodiment, the antibody minimally induces IL-2 and/orIFNγ production from purified T cells under anti-CD3 antibodystimulation, e.g., as measured in the examples described herein, in theabsence of crosslinking of the antibody. Crosslinking of an antibodycontemplated herein includes clustering of the antibody. Methods ofcrosslinking used herein are known in the art. In certain embodiments,the antibody is crosslinked by an agent that dimerizes the Fc region ofthe antibody, e.g., the anti-human IgG (Fab′)₂ as used in the examplesdescribed herein. In certain embodiments, the antibody is crosslinked bycontact with a cell that expresses an Fc receptor that binds to the Fcregion of the antibody (e.g., FcγRIIIa, FcγRIIIb, FcγRIIa, FcγRIIb, orFcγRI). In certain embodiments, the Fc receptor is expressed in acluster on the surface of the cell. In certain embodiments, a ligand ofthe antigen that the antibody binds to is also expressed on the cell. Incertain embodiments, the cell is an antigen-presenting cell (e.g., amacrophage, monocyte, dendritic cell, or B lymphocyte).

In certain embodiments, the ability of the antibody to activate,increase, or promote an activity of CD137 (e.g., human CD137 orcynomolgus CD137) depends on the presence of a ligand of CD137 (e.g.,CD137L (e.g., human CD137 or cynomolgus CD137) or a fragment and/orfusion protein thereof). In certain embodiments, the antibody minimallyincreases or promotes an activity of CD137 (e.g., human CD137 orcynomolgus CD137) in the absence of a ligand of CD137 (e.g., CD137L(e.g., human CD137 or cynomolgus CD137) or a fragment and/or fusionprotein thereof). In certain embodiments, the antibody does notsubstantially increase or promote an activity of CD137 (e.g., humanCD137 or cynomolgus CD137) in the absence of a ligand of CD137 (e.g.,CD137L (e.g., human CD137 or cynomolgus CD137) or a fragment and/orfusion protein thereof). In one embodiment, the antibody minimallyinduces IL-2 and/or IFNγ production from purified T cells under anti-CD3antibody stimulation, e.g., as measured in the examples describedherein. In one embodiment, the antibody minimally induces NF-κBsignaling in a NF-κB reporter cell line, e.g., as measured in theexamples described herein. In certain embodiments, the ability of theantibody to activate, increase, or promote an activity of human CD137positively correlates with the concentration of CD137L. In certainembodiments, the CD137L dependency is observed when the antibody iscrosslinked, e.g., with an anti-human IgG (Fab′)₂ at acrosslinker-to-antibody ratio of about 1:1 to 1:10 (e.g., about 1:1,1:2, 1:3, 1:4, 1:5, or lower).

In specific embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137) and activates a T cell (e.g., a T cell expressinghuman CD137). In certain embodiments, the activated T cell expresses anincreased level (e.g., increased by at least about 1.1 fold, 1.2 fold,1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90fold, or 100 fold) of one or more markers (e.g., perforin, granzyme A,granzyme B, Bcl-X_(L)), optionally wherein the level of the markers canbe measured by flow cytometry. In certain embodiments, the antibodyactivates a T cell (e.g., a T cell expressing human CD137) in thepresence of a ligand of CD137 (e.g., CD137L (e.g., human CD137 orcynomolgus CD137) or a fragment and/or fusion protein thereof). Incertain embodiments, the antibody does not activate a T cell (e.g., a Tcell expressing human CD137) in the absence of a ligand of CD137 (e.g.,CD137L (e.g., human CD137 or cynomolgus CD137) or a fragment and/orfusion protein thereof).

In specific embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137) and increases cytokine production (e.g., IL-2, IFN-γand/or TNF-α) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, asassessed by methods described herein (see the Examples, infra) or knownto one of skill in the art, relative to cytokine production without anyantibody or with an unrelated antibody (e.g., an antibody that does notspecifically bind to CD137 (e.g., human CD137 or cynomolgus CD137)). Inspecific embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137) and increases cytokine production (e.g., IL-2, IFN-γand/or TNF-α) by at least about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold,2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold, as assessed bymethods described herein (see the Examples, infra) or known to one ofskill in the art, relative to cytokine production without any antibodyor with an unrelated antibody (e.g., an antibody that does notspecifically bind to CD137 (e.g., human CD137 or cynomolgus CD137)). Incertain embodiments, the antibody increases cytokine production (e.g.,IL-2, IFN-γ and/or TNF-α) in the presence of a ligand of CD137 (e.g.,CD137L (e.g., human CD137 or cynomolgus CD137) or a fragment and/orfusion protein thereof). In certain embodiments, in the absence of aligand of CD137 (e.g., CD137L (e.g., human CD137 or cynomolgus CD137) ora fragment and/or fusion protein thereof), the antibody does notincrease cytokine production (e.g., IL-2, IFN-γ and/or TNF-α) by morethan 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30%, relative tocytokine production without any antibody or with an unrelated antibody(e.g., an antibody that does not specifically bind to CD137 (e.g., humanCD137 or cynomolgus CD137)).

In specific embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD137 (e.g., human CD137 orcynomolgus CD137) and which either alone or in combination with ananti-PD-1 antibody (e.g., pembrolizumab or nivolumab), increases IL-2and/or IFNγ production in human peripheral blood mononuclear cells(PBMCs) in response to Staphylococcus Enterotoxin A (SEA) stimulation byat least about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold,3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70fold, 80 fold, 90 fold, or 100 fold, as assessed by methods describedherein (see the Examples, infra) or known to one of skill in the art,relative to IL-2 and/or IFNγ production without any antibody or with anunrelated antibody (e.g., an antibody that does not specifically bind toCD137 (e.g., human CD137 or cynomolgus CD137)). In certain embodiments,human peripheral blood mononuclear cells (PBMCs) stimulated withStaphylococcus Enterotoxin A (SEA) in the presence of an antibodydescribed herein, which specifically binds to CD137 (e.g., human CD137or cynomolgus CD137), have increased IL-2 and/or IFNγ production by atleast about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70fold, 80 fold, 90 fold, or 100 fold, relative to IL-2 and/or IFNγproduction from PBMCs only stimulated with SEA without any antibody orwith an unrelated antibody (e.g., an antibody that does not specificallybind to CD137 (e.g., human CD137 or cynomolgus CD137)), as assessed bymethods described herein (see the Examples, infra) or known to one ofskill in the art. In certain embodiments, the antibody increases IL-2and/or IFNγ production in human PBMCs in response to SEA stimulation inthe presence of a ligand of CD137 (e.g., CD137L (e.g., human CD137 orcynomolgus CD137) or a fragment and/or fusion protein thereof). Incertain embodiments, in the absence of a ligand of CD137 (e.g., CD137L(e.g., human CD137 or cynomolgus CD137) or a fragment and/or fusionprotein thereof), the antibody does not increase IL-2 and/or IFNγproduction in human PBMCs in response to SEA stimulation by more than1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30%, relative to cytokineproduction without any antibody or with an unrelated antibody (e.g., anantibody that does not specifically bind to CD137 (e.g., human CD137 orcynomolgus CD137)).

5.3 Pharmaceutical Compositions

Provided herein are compositions comprising an anti-CD137 (e.g., humanCD137 or cynomolgus CD137) antibody disclosed herein having the desireddegree of purity in a physiologically acceptable carrier, excipient orstabilizer (see, e.g., Remington's Pharmaceutical Sciences (1990) MackPublishing Co., Easton, Pa.). Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

In a specific embodiment, pharmaceutical compositions comprise ananti-CD137 (e.g., human CD137 or cynomolgus CD137) antibody disclosedherein, and optionally one or more additional prophylactic ortherapeutic agents, in a pharmaceutically acceptable carrier. In aspecific embodiment, pharmaceutical compositions comprise an effectiveamount of an antibody described herein, and optionally one or moreadditional prophylactic or therapeutic agents, in a pharmaceuticallyacceptable carrier. In certain embodiments, the antibody is the onlyactive ingredient included in the pharmaceutical composition.Pharmaceutical compositions described herein can be useful in increasingor promoting CD137 (e.g., human CD137 or cynomolgus CD137) activity andtreating a condition, such as cancer or an infectious disease. In oneembodiment, the present invention relates to a pharmaceuticalcomposition of the present invention comprising an anti-CD137 antibodyof the present invention for use as a medicament. In another embodiment,the present invention relates to a pharmaceutical composition of thepresent invention for use in a method for the treatment of cancer or aninfectious disease.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances. Examples ofaqueous vehicles include Sodium Chloride Injection, Ringers Injection,Isotonic Dextrose Injection, Sterile Water Injection, Dextrose andLactated Ringers Injection. Nonaqueous parenteral vehicles include fixedoils of vegetable origin, cottonseed oil, corn oil, sesame oil andpeanut oil. Antimicrobial agents in bacteriostatic or fungistaticconcentrations can be added to parenteral preparations packaged inmultiple-dose containers which include phenols or cresols, mercurials,benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acidesters, thimerosal, benzalkonium chloride and benzethonium chloride.Isotonic agents include sodium chloride and dextrose. Buffers includephosphate and citrate. Antioxidants include sodium bisulfate. Localanesthetics include procaine hydrochloride. Suspending and dispersingagents include sodium carboxymethylcelluose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifying agents includePolysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metalions includes EDTA. Pharmaceutical carriers also include ethyl alcohol,polyethylene glycol and propylene glycol for water miscible vehicles;and sodium hydroxide, hydrochloric acid, citric acid or lactic acid forpH adjustment.

A pharmaceutical composition may be formulated for any route ofadministration to a subject. Specific examples of routes ofadministration include intranasal, oral, pulmonary, transdermal,intradermal, and parenteral. Parenteral administration, characterized byeither subcutaneous, intramuscular or intravenous injection, is alsocontemplated herein. Injectables can be prepared in conventional forms,either as liquid solutions or suspensions, solid forms suitable forsolution or suspension in liquid prior to injection, or as emulsions.The injectables, solutions and emulsions also contain one or moreexcipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered can also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Preparations for parenteral administration of an antibody includesterile solutions ready for injection, sterile dry soluble products,such as lyophilized powders, ready to be combined with a solvent justprior to use, including hypodermic tablets, sterile suspensions readyfor injection, sterile dry insoluble products ready to be combined witha vehicle just prior to use and sterile emulsions. The solutions may beeither aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Topical mixtures comprising an antibody are prepared as described forthe local and systemic administration. The resulting mixture can be asolution, suspension, emulsions or the like and can be formulated ascreams, gels, ointments, emulsions, solutions, elixirs, lotions,suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays,suppositories, bandages, dermal patches or any other formulationssuitable for topical administration.

An anti-CD137 (e.g., human CD137 or cynomolgus CD137) antibody disclosedherein can be formulated as an aerosol for topical application, such asby inhalation (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209 and4,364,923, which describe aerosols for delivery of a steroid useful fortreatment of inflammatory diseases, particularly asthma and are hereinincorporated by reference in their entireties). These formulations foradministration to the respiratory tract can be in the form of an aerosolor solution for a nebulizer, or as a microtine powder for insufflations,alone or in combination with an inert carrier such as lactose. In such acase, the particles of the formulation will, in one embodiment, havediameters of less than 50 microns, in one embodiment less than 10microns.

An anti-CD137 (e.g., human CD137 or cynomolgus CD137) antibody disclosedherein can be formulated for local or topical application, such as fortopical application to the skin and mucous membranes, such as in theeye, in the form of gels, creams, and lotions and for application to theeye or for intracisternal or intraspinal application. Topicaladministration is contemplated for transdermal delivery and also foradministration to the eyes or mucosa, or for inhalation therapies. Nasalsolutions of the antibody alone or in combination with otherpharmaceutically acceptable excipients can also be administered.

Transdermal patches, including iontophoretic and electrophoreticdevices, are well known to those of skill in the art, and can be used toadminister an antibody. For example, such patches are disclosed in U.S.Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975,6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, all of whichare herein incorporated by reference in their entireties.

In certain embodiments, a pharmaceutical composition comprising anantibody described herein is a lyophilized powder, which can bereconstituted for administration as solutions, emulsions and othermixtures. It may also be reconstituted and formulated as solids or gels.The lyophilized powder is prepared by dissolving an antibody describedherein, or a pharmaceutically acceptable derivative thereof, in asuitable solvent. In certain embodiments, the lyophilized powder issterile. The solvent may contain an excipient which improves thestability or other pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that may beused include, but are not limited to, dextrose, sorbitol, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent may also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at, inone embodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. In oneembodiment, the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage or multipledosages of the compound. The lyophilized powder can be stored underappropriate conditions, such as at about 4° C. to room temperature.Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder is added to sterile water orother suitable carrier. The precise amount depends upon the selectedcompound. Such amount can be empirically determined.

The anti-CD137 (e.g., human CD137 or cynomolgus CD137) antibodiesdisclosed herein and other compositions provided herein can also beformulated to be targeted to a particular tissue, receptor, or otherarea of the body of the subject to be treated. Many such targetingmethods are well known to those of skill in the art. All such targetingmethods are contemplated herein for use in the instant compositions. Fornon-limiting examples of targeting methods, see, e.g., U.S. Pat. Nos.6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570,6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534,5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542 and 5,709,874, allof which are herein incorporated by reference in their entireties. In aspecific embodiment, an antibody described herein is targeted to atumor.

The compositions to be used for in vivo administration can be sterile.This is readily accomplished by filtration through, e.g., sterilefiltration membranes.

5.4 Methods of Use and Uses

In another aspect, the instant disclosure provides a method of treatinga subject using the anti-CD137 (e.g., human CD137 or cynomolgus CD137)antibodies disclosed herein. Any disease or disorder in a subject thatwould benefit from increase of CD137 (e.g., human CD137 or cynomolgusCD137) function can be treated using the anti-CD137 (e.g., human CD137or cynomolgus CD137) antibodies disclosed herein. The anti-CD137 (e.g.,human CD137) antibodies disclosed herein are particularly useful forinhibiting immune system tolerance to tumors, and accordingly can beused as an immunotherapy for subjects with cancer. For example, incertain embodiments, the instant disclosure provides a method ofincreasing T cell activation in response to an antigen in a subject, themethod comprising administering to the subject an effective amount of ananti-CD137 (e.g., human CD137 or cynomolgus CD137) antibody orpharmaceutical composition thereof, as disclosed herein. In certainembodiments, the instant disclosure provides a method of treating cancerin a subject, the method comprising administering to the subject aneffective amount of the antibody or pharmaceutical composition, asdisclosed herein.

Cancers that can be treated with the anti-CD137 (e.g., human CD137 orcynomolgus CD137) antibodies or pharmaceutical compositions disclosedherein include, without limitation, a solid tumor, a hematologicalcancer (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma), anda metastatic lesion. In one embodiment, the cancer is a solid tumor.Examples of solid tumors include malignancies, e.g., sarcomas andcarcinomas, e.g., adenocarcinomas of the various organ systems, such asthose affecting the lung, breast, ovarian, lymphoid, gastrointestinal(e.g., colon), anal, genitals and genitourinary tract (e.g., renal,urothelial, bladder cells, prostate), pharynx, CNS (e.g., brain, neuralor glial cells), head and neck, skin (e.g., melanoma), and pancreas, aswell as adenocarcinomas which include malignancies such as coloncancers, rectal cancer, renal-cell carcinoma, liver cancer, lung cancer(e.g., non-small cell lung cancer or small cell lung cancer), cancer ofthe small intestine and cancer of the esophagus. The cancer may be at anearly, intermediate, late stage or metastatic cancer.

In one embodiment, the cancer is chosen from lung cancer (e.g., lungadenocarcinoma or non-small cell lung cancer (NSCLC) (e.g., NSCLC withsquamous and/or non-squamous histology, or NSCLC adenocarcinoma)),melanoma (e.g., an advanced melanoma), renal cancer (e.g., a renal cellcarcinoma), liver cancer (e.g., hepatocellular carcinoma), myeloma(e.g., a multiple myeloma), a prostate cancer, a breast cancer (e.g., abreast cancer that does not express one, two or all of estrogenreceptor, progesterone receptor, or Her2/neu, e.g., a triple negativebreast cancer), an ovarian cancer, a colorectal cancer, a pancreaticcancer, a head and neck cancer (e.g., head and neck squamous cellcarcinoma (HNSCC), anal cancer, gastro-esophageal cancer (e.g.,esophageal squamous cell carcinoma), mesothelioma, nasopharyngealcancer, thyroid cancer, cervical cancer, epithelial cancer, peritonealcancer, or a lymphoproliferative disease (e.g., a post-transplantlymphoproliferative disease). In a specific embodiment, the cancer is acervical cancer.

In one embodiment, the cancer is a hematological cancer, for example, aleukemia, a lymphoma, or a myeloma. In one embodiment, the cancer is aleukemia, for example, acute lymphoblastic leukemia (ALL), acutemyelogenous leukemia (AML), acute myeloblastic leukemia (AML), chroniclymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronicmyeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), chroniclymphocytic leukemia (CLL), or hairy cell leukemia. In one embodiment,the cancer is a lymphoma, for example, B cell lymphoma, diffuse largeB-cell lymphoma (DLBCL), activated B-cell like (ABC) diffuse large Bcell lymphoma, germinal center B cell (GCB) diffuse large B celllymphoma, mantle cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma,relapsed non-Hodgkin lymphoma, refractory non-Hodgkin lymphoma,recurrent follicular non-Hodgkin lymphoma, Burkitt lymphoma, smalllymphocytic lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma,or extranodal marginal zone lymphoma. In one embodiment the cancer is amyeloma, for example, multiple myeloma.

In another embodiment, the cancer is chosen from a carcinoma (e.g.,advanced or metastatic carcinoma), melanoma or a lung carcinoma, e.g., anon-small cell lung carcinoma.

In one embodiment, the cancer is a lung cancer, e.g., a lungadenocarcinoma, non-small cell lung cancer or small cell lung cancer.

In one embodiment, the cancer is a melanoma, e.g., an advanced melanoma.In one embodiment, the cancer is an advanced or unresectable melanomathat does not respond to other therapies. In other embodiments, thecancer is a melanoma with a BRAF mutation (e.g., a BRAF V600 mutation).In yet other embodiments, the anti-CD137 (e.g., human CD137 orcynomolgus CD137) antibody or pharmaceutical composition disclosedherein is administered after treatment with an anti-CTLA-4 antibody(e.g., ipilimumab) with or without a BRAF inhibitor (e.g., vemurafenibor dabrafenib).

In another embodiment, the cancer is a hepatocarcinoma, e.g., anadvanced hepatocarcinoma, with or without a viral infection, e.g., achronic viral hepatitis.

In another embodiment, the cancer is a prostate cancer, e.g., anadvanced prostate cancer.

In yet another embodiment, the cancer is a myeloma, e.g., multiplemyeloma.

In yet another embodiment, the cancer is a renal cancer, e.g., a renalcell carcinoma (RCC) (e.g., a metastatic RCC, clear cell renal cellcarcinoma (CCRCC) or kidney papillary cell carcinoma).

In yet another embodiment, the cancer is chosen from a lung cancer, amelanoma, a renal cancer, a breast cancer, a colorectal cancer, aleukemia, or a metastatic lesion of the cancer.

In certain embodiments, the instant disclosure provides a method ofpreventing or treating an infectious disease in a subject, the methodcomprising administering to the subject an effective amount of ananti-CD137 (e.g., human CD137 or cynomolgus CD137) antibody orpharmaceutical composition thereof, as disclosed herein. In oneembodiment, provided herein are methods for preventing and/or treatingan infection (e.g., a viral infection, a bacterial infection, a fungalinfection, a protozoal infection, or a parasitic infection). Theinfection prevented and/or treated in accordance with the methods can becaused by an infectious agent identified herein. In a specificembodiment, an anti-CD137 (e.g., human CD137 or cynomolgus CD137)antibody described herein or a composition thereof is the only activeagent administered to a subject. In certain embodiments, an anti-CD137(e.g., human CD137 or cynomolgus CD137) antibody described herein or acomposition thereof is used in combination with anti-infectiveinterventions (e.g., antivirals, antibacterials, antifungals, oranti-helminthics) for the treatment of infectious diseases. Therefore,in a one embodiment, the present invention relates to an antibody and/orpharmaceutical composition of the present invention for use in a methodof preventing and/or treating an infectious disease, optionally whereinthe antibody or pharmaceutical composition is the only active agentadministered to a subject, or wherein the antibody or pharmaceuticalcomposition is used in combination with anti-infective interventions.

Infectious diseases that can be treated and/or prevented by anti-CD137(e.g., human CD137 or cynomolgus CD137) antibodies or pharmaceuticalcompositions disclosed herein are caused by infectious agents includingbut not limited to bacteria, parasites, fungi, protozae, and viruses. Ina specific embodiment, the infectious disease treated and/or preventedby anti-CD137 (e.g., human CD137 or cynomolgus CD137) antibodies orpharmaceutical compositions disclosed herein is caused by a virus. Viraldiseases or viral infections that can be prevented and/or treated inaccordance with the methods described herein include, but are notlimited to, those caused by hepatitis type A, hepatitis type B,hepatitis type C, influenza (e.g., influenza A or influenza B),varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplextype II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus,mumps virus, measles virus, rubella virus, polio virus, small pox,Epstein Barr virus, human immunodeficiency virus type I (HIV-I), humanimmunodeficiency virus type II (HIV-II), and agents of viral diseasessuch as viral meningitis, encephalitis, dengue or small pox.

Bacterial infections that can be prevented and/or treated includeinfections caused by Escherichia coli, Klebsiella pneumoniae,Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris,Staphylococcus viridans, and Pseudomonas aeruginosa. Bacterial diseasescaused by bacteria (e.g., Escherichia coli, Klebsiella pneumoniae,Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris,Staphylococcus viridans, and Pseudomonas aeruginosa) that can beprevented and/or treated in accordance with the methods described hereininclude, but are not limited to, Mycobacteria rickettsia, Mycoplasma,Neisseria, S. pneumonia, Borrelia burgdorferi (Lyme disease), Bacillusantracis (anthrax), tetanus, Streptococcus, Staphylococcus,mycobacterium, pertissus, cholera, plague, diptheria, chlamydia, S.aureus and legionella.

Protozoal diseases or protozoal infections caused by protozoa that canbe prevented and/or treated in accordance with the methods describedherein include, but are not limited to, Leishmania, Coccidiosis,Trypanosoma schistosoma or malaria. Parasitic diseases or parasiticinfections caused by parasites that can be prevented and/or treated inaccordance with the methods described herein include, but are notlimited to, Chlamydia and Rickettsia.

Fungal diseases or fungal infections that can be prevented and/ortreated in accordance with the methods described herein include, but arenot limited to, those caused by Candida infections, zygomycosis, Candidamastitis, progressive disseminated trichosporonosis with latenttrichosporonemia, disseminated candidiasis, pulmonaryparacoccidioidomycosis, pulmonary aspergillosis, Pneumocystis cariniipneumonia, cryptococcal meningitis, coccidioidal meningoencephalitis andcerebrospinal vasculitis, Aspergillus niger infection, Fusariumkeratitis, paranasal sinus mycoses, Aspergillus fumigatus endocarditis,tibial dyschondroplasia, Candida glabrata vaginitis, oropharyngealcandidiasis, X-linked chronic granulomatous disease, tinea pedis,cutaneous candidiasis, mycotic placentitis, disseminatedtrichosporonosis, allergic bronchopulmonary aspergillosis, mycotickeratitis, Cryptococcus neoformans infection, fungal peritonitis,Curvularia geniculata infection, staphylococcal endophthalmitis,sporotrichosis, and dermatophytosis.

In certain embodiments, these methods further comprise administering anadditional therapeutic agent to the subject. In certain embodiments, theadditional therapeutic agent is a chemotherapeutic, a radiotherapeutic,or a checkpoint targeting agent. In certain embodiments, thechemotherapeutic agent is a hypomethylating agent (e.g., azacitidine).In certain embodiments, the checkpoint targeting agent is selected fromthe group consisting of an antagonist anti-CTLA-4 antibody, anantagonist anti-PD-L1 antibody, an antagonist anti-PD-L2 antibody, anantagonist anti-PD-1 antibody, an antagonist anti-TIM-3 antibody, anantagonist anti-LAG-3 antibody, an antagonist anti-VISTA antibody, anantagonist anti-CD96 antibody, an antagonist anti-CEACAM1 antibody, anantibody anti-TIGIT antibody, an agonist anti-GITR antibody, and anagonist anti-OX40 antibody.

In one embodiment, the present invention relates to an antibody and/orpharmaceutical composition of the present invention for use in a methodof the present invention, wherein the method further comprisesadministering an additional therapeutic agent to the subject. In oneembodiment, the present invention relates to (a) an antibody and/orpharmaceutical composition of the present invention and (b) anadditional therapeutic agent for use as a medicament. In one embodiment,the present invention relates to (a) an antibody and/or pharmaceuticalcomposition of the present invention, and (b) an additional therapeuticagent for use in a method for the treatment of cancer. In a furtherembodiment, the present invention relates to a pharmaceuticalcomposition, kit or kit-of-parts comprising (a) an antibody and/orpharmaceutical composition of the present invention and (b) anadditional therapeutic agent. In one embodiment, the additionaltherapeutic agent is a chemotherapeutic, a radiotherapeutic, or acheckpoint targeting agent.

In certain embodiments, an anti-PD-1 antibody is used in methodsdisclosed herein. In certain embodiments, the anti-PD-1 antibody isnivolumab, also known as BMS-936558 or MDX1106, developed byBristol-Myers Squibb. In certain embodiments, the anti-PD-1 antibody ispembrolizumab, also known as lambrolizumab or MK-3475, developed byMerck & Co. In certain embodiments, the anti-PD-1 antibody ispidilizumab, also known as CT-011, developed by CureTech. In certainembodiments, the anti-PD-1 antibody is MEDI0680, also known as AMP-514,developed by Medimmune. In certain embodiments, the anti-PD-1 antibodyis PDR001 developed by Novartis Pharmaceuticals. In certain embodiments,the anti-PD-1 antibody is REGN2810 developed by RegeneronPharmaceuticals. In certain embodiments, the anti-PD-1 antibody isPF-06801591 developed by Pfizer. In certain embodiments, the anti-PD-1antibody is BGB-A317 developed by BeiGene. In certain embodiments, theanti-PD-1 antibody is TSR-042 developed by AnaptysBio and Tesaro. Incertain embodiments, the anti-PD-1 antibody is SHR-1210 developed byHengrui.

Further non-limiting examples of anti-PD-1 antibodies that may be usedin treatment methods disclosed herein are disclosed in the followingpatents and patent applications, all of which are herein incorporated byreference in their entireties for all purposes: U.S. Pat. Nos.6,808,710; 7,332,582; 7,488,802; 8,008,449; 8,114,845; 8,168,757;8,354,509; 8,686,119; 8,735,553; 8,747,847; 8,779,105; 8,927,697;8,993,731; 9,102,727; 9,205,148; U.S. Publication No. US 2013/0202623A1; U.S. Publication No. US 2013/0291136 A1; U.S. Publication No. US2014/0044738 A1; U.S. Publication No. US 2014/0356363 A1; U.S.Publication No. US 2016/0075783 A1; and PCT Publication No. WO2013/033091 A1; PCT Publication No. WO 2015/036394 A1; PCT PublicationNo. WO 2014/179664 A2; PCT Publication No. WO 2014/209804 A1; PCTPublication No. WO 2014/206107 A1; PCT Publication No. WO 2015/058573A1; PCT Publication No. WO 2015/085847 A1; PCT Publication No. WO2015/200119 A1; PCT Publication No. WO 2016/015685 A1; and PCTPublication No. WO 2016/020856 A1.

In certain embodiments, an anti-PD-L1 antibody is used in methodsdisclosed herein. In certain embodiments, the anti-PD-L1 antibody isatezolizumab developed by Genentech. In certain embodiments, theanti-PD-L1 antibody is durvalumab developed by AstraZeneca, Celgene andMedimmune. In certain embodiments, the anti-PD-L1 antibody is avelumab,also known as MSB0010718C, developed by Merck Serono and Pfizer. Incertain embodiments, the anti-PD-L1 antibody is MDX-1105 developed byBristol-Myers Squibb. In certain embodiments, the anti-PD-L1 antibody isAMP-224 developed by Amplimmune and GSK.

Non-limiting examples of anti-PD-L1 antibodies that may be used intreatment methods disclosed herein are disclosed in the followingpatents and patent applications, all of which are herein incorporated byreference in their entireties for all purposes: U.S. Pat. Nos.7,943,743; 8,168,179; 8,217,149; 8,552,154; 8,779,108; 8,981,063;9,175,082; U.S. Publication No. US 2010/0203056 A1; U.S. Publication No.US 2003/0232323 A1; U.S. Publication No. US 2013/0323249 A1; U.S.Publication No. US 2014/0341917 A1; U.S. Publication No. US 2014/0044738A1; U.S. Publication No. US 2015/0203580 A1; U.S. Publication No. US2015/0225483 A1; U.S. Publication No. US 2015/0346208 A1; U.S.Publication No. US 2015/0355184 A1; and PCT Publication No. WO2014/100079 A1; PCT Publication No. WO 2014/022758 A1; PCT PublicationNo. WO 2014/055897 A2; PCT Publication No. WO 2015/061668 A1; PCTPublication No. WO 2015/109124 A1; PCT Publication No. WO 2015/195163A1; PCT Publication No. WO 2016/000619 A1; and PCT Publication No. WO2016/030350 A1.

In certain embodiments, an anti-CD137 (e.g., human CD137 or cynomolgusCD137) antibody disclosed herein is administered to a subject incombination with a compound that targets an immunomodulatory enzyme(s)such as IDO (indoleamine-(2,3)-dioxygenase) and/or TDO (tryptophan2,3-dioxygenase). Therefore, in one embodiment, the additionaltherapeutic agent is a compound that targets an immunomodulatoryenzyme(s), such as an inhibitor of indoleamine-(2,3)-dioxygenase (IDO).In certain embodiments, such compound is selected from the groupconsisting of epacadostat (Incyte Corp; see, e.g., WO 2010/005958 whichis herein incorporated by reference in its entirety), F001287 (FlexusBiosciences/Bristol-Myers Squibb), indoximod (NewLink Genetics), andNLG919 (NewLink Genetics). In one embodiment, the compound isepacadostat. In another embodiment, the compound is F001287. In anotherembodiment, the compound is indoximod. In another embodiment, thecompound is NLG919. In a specific embodiment, an anti-CD137 (e.g., humanCD137) antibody disclosed herein is administered to a subject incombination with an IDO inhibitor for treating cancer. The IDO inhibitoras described herein for use in treating cancer is present in a soliddosage form of a pharmaceutical composition such as a tablet, a pill ora capsule, wherein the pharmaceutical composition includes an IDOinhibitor and a pharmaceutically acceptable excipient. As such, theantibody as described herein and the IDO inhibitor as described hereincan be administered separately, sequentially or concurrently as separatedosage forms. In one embodiment, the antibody is administeredparenterally, and the IDO inhibitor is administered orally. Inparticular embodiments, the inhibitor is selected from the groupconsisting of epacadostat (Incyte Corporation), F001287 (FlexusBiosciences/Bristol-Myers Squibb), indoximod (NewLink Genetics), andNLG919 (NewLink Genetics). Epacadostat has been described in PCTPublication No. WO 2010/005958, which is herein incorporated byreference in its entirety for all purposes. In one embodiment, theinhibitor is epacadostat. In another embodiment, the inhibitor isF001287. In another embodiment, the inhibitor is indoximod. In anotherembodiment, the inhibitor is NLG919.

In certain embodiments, an anti-CD137 (e.g., human CD137 or cynomolgusCD137) antibody disclosed herein is administered to a subject incombination with a vaccine. The vaccine can be, e.g., a peptide vaccine,a DNA vaccine, or an RNA vaccine. In certain embodiments, the vaccine isa heat shock protein based tumor vaccine or a heat shock protein basedpathogen vaccine. In a specific embodiment, an anti-CD137 (e.g., humanCD137 or cynomolgus CD137) antibody disclosed herein is administered toa subject in combination with a heat shock protein based tumor-vaccine.Heat shock proteins (HSPs) are a family of highly conserved proteinsfound ubiquitously across all species. Their expression can bepowerfully induced to much higher levels as a result of heat shock orother forms of stress, including exposure to toxins, oxidative stress orglucose deprivation. Five families have been classified according tomolecular weight: HSP-110, -90, -70, -60 and -28. HSPs deliverimmunogenic peptides through the cross-presentation pathway in antigenpresenting cells (APCs) such as macrophages and dendritic cells (DCs),leading to T cell activation. HSPs function as chaperone carriers oftumor-associated antigenic peptides forming complexes able to inducetumor-specific immunity. Upon release from dying tumor cells, theHSP-antigen complexes are taken up by antigen-presenting cells (APCs)wherein the antigens are processed into peptides that bind MHC class Iand class II molecules leading to the activation of anti-tumor CD8+ andCD4+ T cells. The immunity elicited by HSP complexes derived from tumorpreparations is specifically directed against the unique antigenicpeptide repertoire expressed by the cancer of each subject. Therefore,in one embodiment, the present invention relates to (a) an antibodyand/or pharmaceutical composition of the present invention and (b) avaccine for use as a medicament, for example for use in a method for thetreatment of cancer. In one embodiment, the present invention relates toa pharmaceutical composition, kit or kit-of-parts comprising (a) anantibody and/or pharmaceutical composition of the present invention and(b) a vaccine. In one embodiment, the vaccine is a heat shock proteinbased tumor vaccine. In one embodiment, the vaccine is a heat shockprotein based pathogen vaccine. In certain embodiments, the vaccine isas described in WO 2016/183486, incorporated herein by reference in itsentirety.

A heat shock protein peptide complex (HSPPC) is a protein peptidecomplex consisting of a heat shock protein non-covalently complexed withantigenic peptides. HSPPCs elicit both innate and adaptive immuneresponses. In a specific embodiment, the antigenic peptide(s) displaysantigenicity for the cancer being treated. HSPPCs are efficiently seizedby APCs via membrane receptors (mainly CD91) or by binding to Toll-likereceptors. HSPPC internalization results in functional maturation of theAPCs with chemokine and cytokine production leading to activation ofnatural killer cells (NK), monocytes and Th1 and Th-2-mediated immuneresponses. In certain embodiments, HSPPCs used in methods disclosedherein comprise one or more heat shock proteins from the hsp60, hsp70,or hsp90 family of stress proteins complexed with antigenic peptides. Incertain embodiments, HSPPCs comprise hsc70, hsp70, hsp90, hsp110,grp170, gp96, calreticulin, or combinations of two or more thereof.

In a specific embodiment, the heat shock protein peptide complex (HSPPC)comprises recombinant heat shock proteins (e.g., hsp70 or hsc70) or apeptide-binding domain thereof complexed with recombinant antigenicpeptides. Recombinant heat shock proteins can be produced by recombinantDNA technology, for example, using human hsc70 sequence as described inDworniczak and Mirault, Nucleic Acids Res. 15:5181-5197 (1987) andGenBank accession no. P11142 and/or Y00371, each of which isincorporated herein by reference in its entirety. In certainembodiments, Hsp70 sequences are as described in Hunt and Morimoto Proc.Natl. Acad. Sci. U.S.A. 82 (19), 6455-6459 (1985) and GenBank accessionno. P0DMV8 and/or M11717, each of which is incorporated herein byreference in its entirety. Antigenic peptides can also be prepared byrecombinant DNA methods known in the art.

In certain embodiments, the antigenic peptides comprise a modified aminoacid. In certain embodiments, the modified amino acid comprises apost-translational modification. In certain embodiments, the modifiedamino acid comprises a mimetic of a post-translational modification. Incertain embodiments, the modified amino acid is a Tyr, Ser, Thr, Arg,Lys, or His that has been phosphorylated on a side chain hydroxyl oramine. In certain embodiments, the modified amino acid is a mimetic of aTyr, Ser, Thr, Arg, Lys, or His amino acid that has been phosphorylatedon a side chain hydroxyl or amine.

In a specific embodiment, an anti-CD137 (e.g., human CD137 or cynomolgusCD137) antibody disclosed herein is administered to a subject incombination with a heat shock protein peptide complex (HSPPC), e.g.,heat shock protein peptide complex-96 (HSPPC-96), to treat cancer.HSPPC-96 comprises a 96 kDa heat shock protein (Hsp), gp96, complexed toantigenic peptides. HSPPC-96 is a cancer immunotherapy manufactured froma subject's tumor and contains the cancer's antigenic “fingerprint.” Incertain embodiments, this fingerprint contains unique antigens that arepresent only in that particular subject's specific cancer cells andinjection of the vaccine is intended to stimulate the subject's immunesystem to recognize and attack any cells with the specific cancerfingerprint. Therefore, in one embodiment, the present invention relatesto an antibody and/or pharmaceutical composition of the presentinvention in combination with a heat shock protein peptide complex(HSPPC) for use as a medicament and/or for use in a method for thetreatment of cancer.

In certain embodiments, the HSPPC, e.g., HSPPC-96, is produced from thetumor tissue of a subject. In a specific embodiment, the HSPPC (e.g.,HSPPC-96) is produced from a tumor of the type of cancer or metastasisthereof being treated. In another specific embodiment, the HSPPC (e.g.,HSPPC-96) is autologous to the subject being treated. In certainembodiments, the tumor tissue is non-necrotic tumor tissue. In certainembodiments, at least 1 gram (e.g., at least 1, at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,or at least 10 grams) of non-necrotic tumor tissue is used to produce avaccine regimen. In certain embodiments, after surgical resection,non-necrotic tumor tissue is frozen prior to use in vaccine preparation.In certain embodiments, the HSPPC, e.g., HSPPC-96, is isolated from thetumor tissue by purification techniques, filtered and prepared for aninjectable vaccine. In certain embodiments, a subject is administered6-12 doses of the HSPPC, e.g., HSPCC-96. In such embodiments, the HSPPC,e.g., HSPPC-96, doses may be administered weekly for the first 4 dosesand then biweekly for the 2-8 additional doses.

Further examples of HSPPCs that may be used in accordance with themethods described herein are disclosed in the following patents andpatent applications, all of which are herein incorporated by referencein their entireties: U.S. Pat. Nos. 6,391,306, 6,383,492, 6,403,095,6,410,026, 6,436,404, 6,447,780, 6,447,781 and 6,610,659.

In certain embodiments, an anti-CD137 (e.g., human CD137 or cynomolgusCD137) antibody disclosed herein is administered to a subject incombination with an adjuvant. Various adjuvants can be used depending onthe treatment context. Non-limiting examples of appropriate adjuvantsinclude, but not limited to, Complete Freund's Adjuvant (CFA),Incomplete Freund's Adjuvant (IFA), montanide ISA (incomplete Seppicadjuvant), the Ribi adjuvant system (RAS), Titer Max, muramyl peptides,Syntex Adjuvant Formulation (SAF), alum (aluminum hydroxide and/oraluminum phosphate), aluminum salt adjuvants, Gerbu® adjuvants,nitrocellulose absorbed antigen, encapsulated or entrapped antigen, 3De-O-acylated monophosphoryl lipid A (3 D-MPL), immunostimulatoryoligonucleotides, toll-like receptor (TLR) ligands, mannan-bindinglectin (MBL) ligands, STING agonists, immuno-stimulating complexes suchas saponins, Quil A, QS-21, QS-7, ISCOMATRIX, and others. Otheradjuvants include CpG oligonucleotides and double stranded RNAmolecules, such as poly(A) and poly(U). Combinations of the aboveadjuvants may also be used. See, e.g., U.S. Pat. Nos. 6,645,495;7,029,678; and 7,858,589, all of which are incorporated herein byreference in their entireties. In one embodiment, the adjuvant usedherein is QS-21 STIMULON.

In certain embodiments, an anti-CD137 (e.g., human CD137 or cynomolgusCD137) antibody disclosed herein is administered to a subject incombination with an additional therapeutic agent comprising a TCR. Incertain embodiments, the additional therapeutic agent is a soluble TCR.In certain embodiments, the additional therapeutic agent is a cellexpressing a TCR. Therefore, in one embodiment, the present inventionrelates to an antibody and/or pharmaceutical composition of the presentinvention in combination with an additional therapeutic agent comprisinga TCR for use as a medicament and/or for use in a method for thetreatment of cancer.

In certain embodiments, an anti-CD137 (e.g., human CD137 or cynomolgusCD137) antibody disclosed herein is administered to a subject incombination with a cell expressing a chimeric antigen receptor (CAR). Incertain embodiments, the cell is a T cell.

In certain embodiments, an anti-CD137 (e.g., human CD137 or cynomolgusCD137) antibody disclosed herein is administered to a subject incombination with a TCR mimic antibody. In certain embodiments, the TCRmimic antibody is an antibody that specifically binds to a peptide-MHCcomplex. For non-limiting examples of TCR mimic antibodies, see, e.g.,U.S. Pat. No. 9,074,000 and U.S. Publication Nos. US 2009/0304679 A1 andUS 2014/0134191 A1, all of which are incorporated herein by reference intheir entireties.

In certain embodiments, an anti-CD137 (e.g., human CD137 or cynomolgusCD137) antibody disclosed herein is administered to a subject incombination with a bispecific T-cell engager (BiTE) (e.g., as describedin WO2005061547A2, which is incorporated by reference herein in itsentirety) and/or a dual-affinity re-targeting antibody (DART) (e.g., asdescribed in WO2012162067A2, which is incorporated by reference hereinin its entirety). In certain embodiments, the BiTE and/or DARTspecifically binds to a tumor-associated antigen (e.g., a polypeptideoverexpressed in a tumor, a polypeptide derived from an oncovirus, apolypeptide comprising a post-translational modification specific to atumor, a polypeptide specifically mutated in a tumor) and a molecule onan effector cell (e.g., CD3 or CD16). In certain embodiments, thetumor-associated antigen is EGFR (e.g., human EGFR), Her2 (e.g., humanHer2), or CD20 (e.g., human CD20).

The anti-CD137 (e.g., human CD137 or cynomolgus CD137) antibody and theadditional therapeutic agent (e.g., chemotherapeutic, radiotherapeutic,checkpoint targeting agent, IDO inhibitor, vaccine, adjuvant, a solubleTCR, a cell expressing a TCR, a cell expressing a chimeric antigenreceptor, and/or a TCR mimic antibody) can be administered separately,sequentially or concurrently as separate dosage forms. In oneembodiment, an anti-CD137 (e.g., human CD137 or cynomolgus CD137)antibody is administered parenterally, and an IDO inhibitor isadministered orally.

An antibody or pharmaceutical composition described herein may bedelivered to a subject by a variety of routes. These include, but arenot limited to, parenteral, intranasal, intratracheal, oral,intradermal, topical, intramuscular, intraperitoneal, transdermal,intravenous, intratumoral, conjunctival, intra-arterial, andsubcutaneous routes. Pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent for use as a spray. In certain embodiments, theantibody or pharmaceutical composition described herein is deliveredsubcutaneously or intravenously. In certain embodiments, the antibody orpharmaceutical composition described herein is deliveredintra-arterially. In certain embodiments, the antibody or pharmaceuticalcomposition described herein is delivered intratumorally. In certainembodiments, the antibody or pharmaceutical composition described hereinis delivered into a tumor draining lymph node.

The amount of an antibody or composition which will be effective in thetreatment and/or prevention of a condition will depend on the nature ofthe disease, and can be determined by standard clinical techniques.

The precise dose to be employed in a composition will also depend on theroute of administration, and the seriousness of the infection or diseasecaused by it, and should be decided according to the judgment of thepractitioner and each subject's circumstances. For example, effectivedoses may also vary depending upon means of administration, target site,physiological state of the patient (including age, body weight andhealth), whether the patient is human or an animal, other medicationsadministered, or whether treatment is prophylactic or therapeutic.Usually, the patient is a human, but non-human mammals, includingtransgenic mammals, can also be treated. Treatment dosages are optimallytitrated to optimize safety and efficacy.

An anti-CD137 (e.g., human CD137 or cynomolgus CD137) antibody describedherein can also be used to assay CD137 (e.g., human CD137 or cynomolgusCD137) protein levels in a biological sample using classicalimmunohistological methods known to those of skill in the art, includingimmunoassays, such as the enzyme linked immunosorbent assay (ELISA),immunoprecipitation, or Western blotting. Suitable antibody assay labelsare known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (¹²⁵I, ¹²¹I) carbon (¹⁴C), sulfur(³⁵S), tritium (³H), indium (¹²¹In), and technetium (⁹⁹Tc); luminescentlabels, such as luminol; and fluorescent labels, such as fluorescein andrhodamine, and biotin. Such labels can be used to label an antibodydescribed herein. Alternatively, a second antibody that recognizes ananti-CD137 (e.g., human CD137 or cynomolgus CD137) antibody describedherein can be labeled and used in combination with an anti-CD137 (e.g.,human CD137 or cynomolgus CD137) antibody to detect CD137 (e.g., humanCD137 or cynomolgus CD137) protein levels. Therefore, in one embodiment,the present invention relates to the use of an antibody of the presentinvention for in vitro detection of CD137 (e.g., human CD137 orcynomolgus CD137) protein in a biological sample. In a furtherembodiment, the present invention relates to the use of an anti-CD137antibody of the invention, for assaying and/or detecting CD137 (e.g.,human CD137 or cynomolgus CD137) protein levels in a biological samplein vitro, optionally wherein the anti-CD137 antibody is conjugated to aradionuclide or detectable label, and/or carries a label describedherein, and/or wherein an immunohistological method is used.

Assaying for the expression level of CD137 (e.g., human CD137 orcynomolgus CD137) protein is intended to include qualitatively orquantitatively measuring or estimating the level of CD137 (e.g., humanCD137 or cynomolgus CD137) protein in a first biological sample eitherdirectly (e.g., by determining or estimating absolute protein level) orrelatively (e.g., by comparing to the disease associated protein levelin a second biological sample). CD137 (e.g., human CD137 or cynomolgusCD137) polypeptide expression level in the first biological sample canbe measured or estimated and compared to a standard CD137 (e.g., humanCD137 or cynomolgus CD137) protein level, the standard being taken, forexample, from a second biological sample obtained from an individual nothaving the disorder or being determined by averaging levels from apopulation of individuals not having the disorder. As will beappreciated in the art, once the “standard” CD137 (e.g., human CD137 orcynomolgus CD137) polypeptide level is known, it can be used repeatedlyas a standard for comparison. Therefore, in a further embodiment, thepresent invention relates to an in vitro method for assaying and/ordetecting CD137 protein levels, for example human CD137 protein levels,in a biological sample, comprising qualitatively or quantitativelymeasuring or estimating the level of CD137 protein, for example of humanCD137 protein, in a biological sample, by an immunohistological method.

As used herein, the term “biological sample” refers to any biologicalsample obtained from a subject, cell line, tissue, or other source ofcells potentially expressing CD137 (e.g., human CD137 or cynomolgusCD137). Methods for obtaining tissue biopsies and body fluids fromanimals (e.g., humans or cynomolgus monkeys) are well known in the art.Biological samples include peripheral mononuclear blood cells.

An anti-CD137 (e.g., human CD137 or cynomolgus CD137) antibody describedherein can be used for prognostic, diagnostic, monitoring and screeningapplications, including in vitro and in vivo applications well known andstandard to the skilled artisan and based on the present description.Prognostic, diagnostic, monitoring and screening assays and kits for invitro assessment and evaluation of immune system status and/or immuneresponse may be utilized to predict, diagnose and monitor to evaluatepatient samples including those known to have or suspected of having animmune system-dysfunction or with regard to an anticipated or desiredimmune system response, antigen response or vaccine response. Theassessment and evaluation of immune system status and/or immune responseis also useful in determining the suitability of a patient for aclinical trial of a drug or for the administration of a particularchemotherapeutic agent, a radiotherapeutic agent, or an antibody,including combinations thereof, versus a different agent or antibody.This type of prognostic and diagnostic monitoring and assessment isalready in practice utilizing antibodies against the HER2 protein inbreast cancer (HercepTest™, Dako) where the assay is also used toevaluate patients for antibody therapy using Herceptin®. In vivoapplications include directed cell therapy and immune system modulationand radio imaging of immune responses. Therefore, in one embodiment, thepresent invention relates to an anti-CD137 antibody and/orpharmaceutical composition of the present invention for use as adiagnostic. In one embodiment, the present invention relates to ananti-CD137 antibody and/or pharmaceutical composition of the presentinvention for use in a method for the prediction, diagnosis and/ormonitoring of a subject having or suspected to have an immunesystem-dysfunction and/or with regard to an anticipated or desiredimmune system response, antigen response or vaccine response. In anotherembodiment, the present invention relates to the use of anti-CD137antibody of the invention, for predicting, diagnosing and/or monitoringof a subject having or suspected to have an immune system-dysfunctionand/or with regard to an anticipated or desired immune system response,antigen response or vaccine response by assaying and/or detecting humanCD137 protein levels in a biological sample of the subject in vitro.

In one embodiment, an anti-CD137 (e.g., human CD137 or cynomolgus CD137)antibody can be used in immunohistochemistry of biopsy samples. In oneembodiment, the method is an in vitro method. In another embodiment, ananti-CD137 (e.g., human CD137 or cynomolgus CD137) antibody can be usedto detect levels of CD137 (e.g., human CD137 or cynomolgus CD137), orlevels of cells which contain CD137 (e.g., human CD137 or cynomolgusCD137) on their membrane surface, the levels of which can then be linkedto certain disease symptoms. Anti-CD137 (e.g., human CD137 or cynomolgusCD137) antibodies described herein may carry a detectable or functionallabel and/or may be conjugated to a radionuclide or detectable label.When fluorescence labels are used, currently available microscopy andfluorescence-activated cell sorter analysis (FACS) or combination ofboth methods procedures known in the art may be utilized to identify andto quantitate the specific binding members. Anti-CD137 (e.g., humanCD137 or cynomolgus CD137) antibodies described herein may carry or maybe conjugated to a fluorescence label. Exemplary fluorescence labelsinclude, for example, reactive and conjugated probes, e.g.,Aminocoumarin, Fluorescein and Texas red, Alexa Fluor dyes, Cy dyes andDyLight dyes. An anti-CD137 (e.g., human CD137 or cynomolgus CD137)antibody may carry or may be conjugated to a radioactive label orradionuclide, such as the isotopes ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co,⁵⁸Co, ⁵⁹Fe, ⁶⁷Cu, ⁹⁰Y, ⁹⁹Te, ¹¹¹In, ¹¹⁷Lu, ¹²¹I, ¹²⁴I, ¹²⁵I, ¹³¹I,¹⁹⁸Au, ²¹¹At, ²¹³Bi, ²²⁵Ac and ¹⁸⁶Re. When radioactive labels are used,currently available counting procedures known in the art may be utilizedto identify and quantitate the specific binding of anti-CD137 (e.g.,human CD137 or cynomolgus CD137) antibody to CD137 (e.g., human CD137 orcynomolgus CD137). In the instance where the label is an enzyme,detection may be accomplished by any of the presently utilizedcolorimetric, spectrophotometric, fluorospectrophotometric, amperometricor gasometric techniques as known in the art. This can be achieved bycontacting a sample or a control sample with an anti-CD137 (e.g., humanCD137 or cynomolgus CD137) antibody under conditions that allow for theformation of a complex between the antibody and CD137 (e.g., human CD137or cynomolgus CD137). Any complexes formed between the antibody andCD137 (e.g., human CD137 or cynomolgus CD137) are detected and comparedin the sample and the control. In light of the specific binding of theantibodies described herein for CD137 (e.g., human CD137 or cynomolgusCD137), the antibodies can be used to specifically detect CD137 (e.g.,human CD137 or cynomolgus CD137) expression on the surface of cells. Theantibodies described herein can also be used to purify CD137 (e.g.,human CD137 or cynomolgus CD137) via immunoaffinity purification. Alsoincluded herein is an assay system which may be prepared in the form ofa test kit, kit, or kit-of-parts for the quantitative analysis of theextent of the presence of, for instance, CD137 (e.g., human CD137 orcynomolgus CD137) or CD137 (e.g., human CD137 or cynomolgus CD137)/CD137(e.g., human CD137 or cynomolgus CD137) ligand complexes. The system,test kit, kit or kit-of-parts may comprise a labeled component, e.g., alabeled antibody, and one or more additional immunochemical reagents.

5.5 Polynucleotides, Vectors and Methods of Producing Anti-CD137Antibodies

In another aspect, provided herein are polynucleotides comprising anucleotide sequence encoding an antibody described herein or a fragmentthereof (e.g., a light chain variable region and/or heavy chain variableregion) that specifically binds to a CD137 (e.g., human CD137 orcynomolgus CD137) antigen, and vectors, e.g., vectors comprising suchpolynucleotides for recombinant expression in host cells (e.g., E. coliand mammalian cells). Provided herein are polynucleotides comprisingnucleotide sequences encoding a heavy and/or light chain of any of theantibodies provided herein, as well as vectors comprising suchpolynucleotide sequences, e.g., expression vectors for their efficientexpression in host cells, e.g., mammalian cells.

As used herein, an “isolated” polynucleotide or nucleic acid molecule isone which is separated from other nucleic acid molecules which arepresent in the natural source (e.g., in a mouse or a human) of thenucleic acid molecule. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. For example, the language “substantially free”includes preparations of polynucleotide or nucleic acid molecule havingless than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (in particular lessthan about 10%) of other material, e.g., cellular material, culturemedium, other nucleic acid molecules, chemical precursors and/or otherchemicals. In a specific embodiment, a nucleic acid molecule(s) encodingan antibody described herein is isolated or purified.

In particular aspects, provided herein are polynucleotides comprisingnucleotide sequences encoding antibodies, which specifically bind to aCD137 (e.g., human CD137 or cynomolgus CD137) polypeptide and comprisesan amino acid sequence as described herein, as well as antibodies whichcompete with such antibodies for binding to a CD137 (e.g., human CD137or cynomolgus CD137) polypeptide (e.g., in a dose-dependent manner), orwhich binds to the same epitope as that of such antibodies.

In certain aspects, provided herein are polynucleotides comprising anucleotide sequence encoding the light chain or heavy chain of anantibody described herein. The polynucleotides can comprise nucleotidesequences encoding a light chain comprising the VL FRs and CDRs ofantibodies described herein (see, e.g., Table 1) or nucleotide sequencesencoding a heavy chain comprising the VH FRs and CDRs of antibodiesdescribed herein (see, e.g., Table 1).

Also provided herein are polynucleotides encoding an anti-CD137 (e.g.,human CD137 or cynomolgus CD137) antibody that are optimized, e.g., bycodon/RNA optimization, replacement with heterologous signal sequences,and elimination of mRNA instability elements. Methods to generateoptimized nucleic acids encoding an anti-CD137 (e.g., human CD137 orcynomolgus CD137) antibody or a fragment thereof (e.g., light chain,heavy chain, VH domain, or VL domain) for recombinant expression byintroducing codon changes and/or eliminating inhibitory regions in themRNA can be carried out by adapting the optimization methods describedin, e.g., U.S. Pat. Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and6,794,498, accordingly, all of which are herein incorporated byreference in their entireties. For example, potential splice sites andinstability elements (e.g., A/T or A/U rich elements) within the RNA canbe mutated without altering the amino acids encoded by the nucleic acidsequences to increase stability of the RNA for recombinant expression.The alterations utilize the degeneracy of the genetic code, e.g., usingan alternative codon for an identical amino acid. In certainembodiments, it can be desirable to alter one or more codons to encode aconservative mutation, e.g., a similar amino acid with similar chemicalstructure and properties and/or function as the original amino acid.Such methods can increase expression of an anti-CD137 (e.g., human CD137or cynomolgus CD137) antibody or fragment thereof by at least 1 fold, 2fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold or more relativeto the expression of an anti-CD137 (e.g., human CD137 or cynomolgusCD137) antibody encoded by polynucleotides that have not been optimized.

In certain embodiments, an optimized polynucleotide sequence encoding ananti-CD137 (e.g., human CD137 or cynomolgus CD137) antibody describedherein or a fragment thereof (e.g., VL domain and/or VH domain) canhybridize to an antisense (e.g., complementary) polynucleotide of anunoptimized polynucleotide sequence encoding an anti-CD137 (e.g., humanCD137 or cynomolgus CD137) antibody described herein or a fragmentthereof (e.g., VL domain and/or VH domain). In specific embodiments, anoptimized nucleotide sequence encoding an anti-CD137 (e.g., human CD137or cynomolgus CD137) antibody described herein or a fragment hybridizesunder high stringency conditions to antisense polynucleotide of anunoptimized polynucleotide sequence encoding an anti-CD137 (e.g., humanCD137 or cynomolgus CD137) antibody described herein or a fragmentthereof. In a specific embodiment, an optimized nucleotide sequenceencoding an anti-CD137 (e.g., human CD137 or cynomolgus CD137) antibodydescribed herein or a fragment thereof hybridizes under high stringency,intermediate or lower stringency hybridization conditions to anantisense polynucleotide of an unoptimized nucleotide sequence encodingan anti-CD137 (e.g., human CD137 or cynomolgus CD137) antibody describedherein or a fragment thereof. Information regarding hybridizationconditions has been described, see, e.g., U.S. Patent ApplicationPublication No. US 2005/0048549 (e.g., paragraphs 72-73), which isherein incorporated by reference in its entirety.

The polynucleotides can be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. Nucleotidesequences encoding antibodies described herein, e.g., antibodiesdescribed in Table 1, and modified versions of these antibodies can bedetermined using methods well known in the art, i.e., nucleotide codonsknown to encode particular amino acids are assembled in such a way togenerate a nucleic acid that encodes the antibody. Such a polynucleotideencoding the antibody can be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier G et al., (1994),BioTechniques 17: 242-6, herein incorporated by reference in itsentirety), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody described hereincan be generated from nucleic acid from a suitable source (e.g., ahybridoma) using methods well known in the art (e.g., PCR and othermolecular cloning methods). For example, PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of a known sequencecan be performed using genomic DNA obtained from hybridoma cellsproducing the antibody of interest. Such PCR amplification methods canbe used to obtain nucleic acids comprising the sequence encoding thelight chain and/or heavy chain of an antibody. Such PCR amplificationmethods can be used to obtain nucleic acids comprising the sequenceencoding the variable light chain region and/or the variable heavy chainregion of an antibody. The amplified nucleic acids can be cloned intovectors for expression in host cells and for further cloning, forexample, to generate chimeric and humanized antibodies.

If a clone containing a nucleic acid encoding a particular antibody isnot available, but the sequence of the antibody molecule is known, anucleic acid encoding the immunoglobulin can be chemically synthesizedor obtained from a suitable source (e.g., an antibody cDNA library or acDNA library generated from, or nucleic acid, preferably poly A+RNA,isolated from, any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody described herein) by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR can then be cloned into replicable cloning vectorsusing any method well known in the art.

DNA encoding anti-CD137 (e.g., human CD137 or cynomolgus CD137)antibodies described herein can be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the anti-CD137 (e.g., human CD137 or cynomolgus CD137)antibodies). Hybridoma cells can serve as a source of such DNA. Onceisolated, the DNA can be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GSSystem™ (Lonza)), or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of anti-CD137 (e.g.,human CD137 or cynomolgus CD137) antibodies in the recombinant hostcells.

To generate whole antibodies, PCR primers including VH or VL nucleotidesequences, a restriction site, and a flanking sequence to protect therestriction site can be used to amplify the VH or VL sequences in scFvclones. Utilizing cloning techniques known to those of skill in the art,the PCR amplified VH domains can be cloned into vectors expressing aheavy chain constant region, e.g., the human gamma 4 constant region,and the PCR amplified VL domains can be cloned into vectors expressing alight chain constant region, e.g., human kappa or lambda constantregions. In certain embodiments, the vectors for expressing the VH or VLdomains comprise an EF-1α promoter, a secretion signal, a cloning sitefor the variable region, constant domains, and a selection marker suchas neomycin. The VH and VL domains can also be cloned into one vectorexpressing the necessary constant regions. The heavy chain conversionvectors and light chain conversion vectors are then co-transfected intocell lines to generate stable or transient cell lines that expressfull-length antibodies, e.g., IgG, using techniques known to those ofskill in the art.

The DNA also can be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe murine sequences, or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide.

Also provided are polynucleotides that hybridize under high stringency,intermediate or lower stringency hybridization conditions topolynucleotides that encode an antibody described herein. In specificembodiments, polynucleotides described herein hybridize under highstringency, intermediate or lower stringency hybridization conditions topolynucleotides encoding a VH domain and/or VL domain provided herein.

Hybridization conditions have been described in the art and are known toone of skill in the art. For example, hybridization under stringentconditions can involve hybridization to filter-bound DNA in 6x sodiumchloride/sodium citrate (SSC) at about 45° C. followed by one or morewashes in 0.2×SSC/0.1% SDS at about 50-65° C.; hybridization underhighly stringent conditions can involve hybridization to filter-boundnucleic acid in 6×SSC at about 45° C. followed by one or more washes in0.1×SSC/0.2% SDS at about 68° C. Hybridization under other stringenthybridization conditions are known to those of skill in the art and havebeen described, see, for example, Ausubel F M et al., eds., (1989)Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York at pages6.3.1-6.3.6 and 2.10.3, which is herein incorporated by reference in itsentirety.

In certain aspects, provided herein are cells (e.g., host cells)expressing (e.g., recombinantly) antibodies described herein whichspecifically bind to CD137 (e.g., human CD137 or cynomolgus CD137) andrelated polynucleotides and expression vectors. Provided herein arevectors (e.g., expression vectors) comprising polynucleotides comprisingnucleotide sequences encoding anti-CD137 (e.g., human CD137 orcynomolgus CD137) antibodies or a fragment for recombinant expression inhost cells, preferably in mammalian cells (e.g., CHO cells). Alsoprovided herein are host cells comprising such vectors for recombinantlyexpressing anti-CD137 (e.g., human CD137 or cynomolgus CD137) antibodiesdescribed herein (e.g., human or humanized antibody). In a particularaspect, provided herein are methods for producing an antibody describedherein, comprising expressing such antibody from a host cell.

Recombinant expression of an antibody described herein (e.g., afull-length antibody, heavy and/or light chain of an antibody, or asingle chain antibody described herein) that specifically binds to CD137(e.g., human CD137 or cynomolgus CD137) generally involves constructionof an expression vector containing a polynucleotide that encodes theantibody. Once a polynucleotide encoding an antibody molecule, heavyand/or light chain of an antibody, or a fragment thereof (e.g., heavyand/or light chain variable regions) described herein has been obtained,the vector for the production of the antibody molecule can be producedby recombinant DNA technology using techniques well known in the art.Thus, methods for preparing a protein by expressing a polynucleotidecontaining an antibody or antibody fragment (e.g., light chain or heavychain) encoding nucleotide sequence are described herein. Methods whichare well known to those skilled in the art can be used to constructexpression vectors containing antibody or antibody fragment (e.g., lightchain or heavy chain) coding sequences and appropriate transcriptionaland translational control signals. These methods include, for example,in vitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. Also provided are replicable vectors comprising anucleotide sequence encoding an antibody molecule described herein, aheavy or light chain of an antibody, a heavy or light chain variableregion of an antibody or a fragment thereof, or a heavy or light chainCDR, operably linked to a promoter. Such vectors can, for example,include the nucleotide sequence encoding the constant region of theantibody molecule (see, e.g., International Publication Nos. WO 86/05807and WO 89/01036; and U.S. Pat. No. 5,122,464, which are hereinincorporated by reference in their entireties) and variable regions ofthe antibody can be cloned into such a vector for expression of theentire heavy, the entire light chain, or both the entire heavy and lightchains.

An expression vector can be transferred to a cell (e.g., host cell) byconventional techniques and the resulting cells can then be cultured byconventional techniques to produce an antibody described herein or afragment thereof. Thus, provided herein are host cells containing apolynucleotide encoding an antibody described herein or fragmentsthereof, or a heavy or light chain thereof, or fragment thereof, or asingle chain antibody described herein, operably linked to a promoterfor expression of such sequences in the host cell. In certainembodiments, for the expression of double-chained antibodies, vectorsencoding both the heavy and light chains, individually, can beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below. In certain embodiments, ahost cell contains a vector comprising a polynucleotide encoding boththe heavy chain and light chain of an antibody described herein, or afragment thereof. In specific embodiments, a host cell contains twodifferent vectors, a first vector comprising a polynucleotide encoding aheavy chain or a heavy chain variable region of an antibody describedherein, or a fragment thereof, and a second vector comprising apolynucleotide encoding a light chain or a light chain variable regionof an antibody described herein, or a fragment thereof. In otherembodiments, a first host cell comprises a first vector comprising apolynucleotide encoding a heavy chain or a heavy chain variable regionof an antibody described herein, or a fragment thereof, and a secondhost cell comprises a second vector comprising a polynucleotide encodinga light chain or a light chain variable region of an antibody describedherein. In specific embodiments, a heavy chain/heavy chain variableregion expressed by a first cell associated with a light chain/lightchain variable region of a second cell to form an anti-CD137 (e.g.,human CD137 or cynomolgus CD137) antibody described herein. In certainembodiments, provided herein is a population of host cells comprisingsuch first host cell and such second host cell.

In a particular embodiment, provided herein is a population of vectorscomprising a first vector comprising a polynucleotide encoding a lightchain/light chain variable region of an anti-CD137 (e.g., human CD137 orcynomolgus CD137) antibody described herein, and a second vectorcomprising a polynucleotide encoding a heavy chain/heavy chain variableregion of an anti-CD137 (e.g., human CD137 or cynomolgus CD137) antibodydescribed herein.

A variety of host-expression vector systems can be utilized to expressantibody molecules described herein (see, e.g., U.S. Pat. No. 5,807,715,which is herein incorporated by reference in its entirety). Suchhost-expression systems represent vehicles by which the coding sequencesof interest can be produced and subsequently purified, but alsorepresent cells which can, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody moleculedescribed herein in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli and B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing antibody coding sequences; yeast(e.g., Saccharomyces pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systems(e.g., green algae such as Chlamydomonas reinhardtii) infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS),CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, andNIH 3T3, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20 andBMT10 cells) harboring recombinant expression constructs containingpromoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). In aspecific embodiment, cells for expressing antibodies described hereinare Chinese hamster ovary (CHO) cells, for example CHO cells from theCHO GS System™ (Lonza). In certain embodiments, the heavy chain and/orlight chain of an antibody produced by a CHO cell may have an N-terminalglutamine or glutamate residue replaced by pyroglutamate. In aparticular embodiment, cells for expressing antibodies described hereinare human cells, e.g., human cell lines. In a specific embodiment, amammalian expression vector is pOptiVEC™ or pcDNA3.3. In a particularembodiment, bacterial cells such as Escherichia coli, or eukaryoticcells (e.g., mammalian cells), especially for the expression of wholerecombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such as CHOcells, in conjunction with a vector such as the major intermediate earlygene promoter element from human cytomegalovirus is an effectiveexpression system for antibodies (Foecking M K & Hofstetter H (1986)Gene 45: 101-5; and Cockett M I et al., (1990) Biotechnology 8(7):662-7, each of which is herein incorporated by reference in itsentirety). In certain embodiments, antibodies described herein areproduced by CHO cells or NS0 cells. In a specific embodiment, theexpression of nucleotide sequences encoding antibodies described hereinwhich specifically bind to CD137 (e.g., human CD137 or cynomolgus CD137)is regulated by a constitutive promoter, inducible promoter or tissuespecific promoter.

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such anantibody is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified can be desirable. Such vectors include, but are not limited to,the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983)EMBO J 2: 1791-1794), in which the antibody coding sequence can beligated individually into the vector in frame with the lac Z codingregion so that a fusion protein is produced; pIN vectors (Inouye S &Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster S M(1989) J Biol Chem 24: 5503-5509); and the like, all of which are hereinincorporated by reference in their entireties. For example, pGEX vectorscan also be used to express foreign polypeptides as fusion proteins withglutathione 5-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption andbinding to matrix glutathione agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetgene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV), for example, can be used as a vector to express foreign genes.The virus grows in Spodoptera frugiperda cells. The antibody codingsequence can be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region Elor E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see Logan J &Shenk T (1984) PNAS 81(12): 3655-9, which is herein incorporated byreference in its entirety). Specific initiation signals can also berequired for efficient translation of inserted antibody codingsequences. These signals include the ATG initiation codon and adjacentsequences. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression can be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see, e.g., Bitter G et al., (1987) Methods Enzymol.153: 516-544, which is herein incorporated by reference in itsentirety).

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, Hela, MDCK, HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murinemyeloma cell line that does not endogenously produce any immunoglobulinchains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst,HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20, BMT10 andHsS78Bst cells. In certain embodiments, anti-CD137 (e.g., human CD137 orcynomolgus CD137) antibodies described herein are produced in mammaliancells, such as CHO cells.

In a specific embodiment, the antibodies described herein have reducedfucose content or no fucose content. Such antibodies can be producedusing techniques known one skilled in the art. For example, theantibodies can be expressed in cells deficient or lacking the ability ofto fucosylate. In a specific example, cell lines with a knockout of bothalleles of α1,6-fucosyltransferase can be used to produce antibodieswith reduced fucose content. The Potelligent® system (Lonza) is anexample of such a system that can be used to produce antibodies withreduced fucose content.

For long-term, high-yield production of recombinant proteins, stableexpression cells can be generated. For example, cell lines which stablyexpress an anti-CD137 (e.g., human CD137 or cynomolgus CD137) antibodydescribed herein can be engineered. In specific embodiments, a cellprovided herein stably expresses a light chain/light chain variableregion and a heavy chain/heavy chain variable region which associate toform an antibody described herein.

In certain aspects, rather than using expression vectors which containviral origins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoter,enhancer, sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. Following the introduction of theforeign DNA/polynucleotide, engineered cells can be allowed to grow for1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which in turn canbe cloned and expanded into cell lines. This method can advantageouslybe used to engineer cell lines which express an anti-CD137 (e.g., humanCD137 or cynomolgus CD137) antibody described herein or a fragmentthereof. Such engineered cell lines can be particularly useful inscreening and evaluation of compositions that interact directly orindirectly with the antibody molecule.

A number of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell11(1): 223-32), hypoxanthineguanine phosphoribosyltransferase (SzybalskaE H & Szybalski W (1962) PNAS 48(12): 2026-2034) and adeninephosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3): 817-23)genes in tk-, hgprt- or aprt-cells, respectively, all of which areherein incorporated by reference in their entireties. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (WiglerM et al., (1980) PNAS 77(6): 3567-70; O'Hare K et al., (1981) PNAS 78:1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo, which confers resistance tothe aminoglycoside G-418 (Wu G Y & Wu C H (1991) Biotherapy 3: 87-95;Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan R C(1993) Science 260: 926-932; and Morgan R A & Anderson W F (1993) AnnRev Biochem 62: 191-217; Nabel G J & Felgner P L (1993) TrendsBiotechnol 11(5): 211-5); and hygro, which confers resistance tohygromycin (Santerre R F et al., (1984) Gene 30(1-3): 147-56), all ofwhich are herein incorporated by reference in their entireties. Methodscommonly known in the art of recombinant DNA technology can be routinelyapplied to select the desired recombinant clone and such methods aredescribed, for example, in Ausubel F M et al., (eds.), Current Protocolsin Molecular Biology, John Wiley & Sons, N Y (1993); Kriegler M, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, N Y(1990); and in Chapters 12 and 13, Dracopoli N C et al., (eds.), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colbère-Garapin F et al., (1981) J Mol Biol 150: 1-14, all of which areherein incorporated by reference in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington C R & Hentschel C C G, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, NewYork, 1987), which is herein incorporated by reference in its entirety).When a marker in the vector system expressing antibody is amplifiable,increase in the level of inhibitor present in culture of host cell willincrease the number of copies of the marker gene. Since the amplifiedregion is associated with the antibody gene, production of the antibodywill also increase (Crouse G F et al., (1983) Mol Cell Biol 3: 257-66,which is herein incorporated by reference in its entirety).

The host cell can be co-transfected with two or more expression vectorsdescribed herein, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors can contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides. Thehost cells can be co-transfected with different amounts of the two ormore expression vectors. For example, host cells can be transfected withany one of the following ratios of a first expression vector and asecond expression vector: about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

Alternatively, a single vector can be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot N.J. (1986) Nature322: 562-565; and Köhler G (1980) PNAS 77: 2197-2199, each of which isherein incorporated by reference in its entirety). The coding sequencesfor the heavy and light chains can comprise cDNA or genomic DNA. Theexpression vector can be monocistronic or multicistronic. Amulticistronic nucleic acid construct can encode 2, 3, 4, 5, 6, 7, 8, 9,10 or more genes/nucleotide sequences, or in the range of 2-5, 5-10, or10-20 genes/nucleotide sequences. For example, a bicistronic nucleicacid construct can comprise, in the following order, a promoter, a firstgene (e.g., heavy chain of an antibody described herein), and a secondgene and (e.g., light chain of an antibody described herein). In such anexpression vector, the transcription of both genes can be driven by thepromoter, whereas the translation of the mRNA from the first gene can beby a cap-dependent scanning mechanism and the translation of the mRNAfrom the second gene can be by a cap-independent mechanism, e.g., by anIRES.

Once an antibody molecule described herein has been produced byrecombinant expression, it can be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theantibodies described herein can be fused to heterologous polypeptidesequences described herein or otherwise known in the art to facilitatepurification.

In specific embodiments, an antibody described herein is isolated orpurified. Generally, an isolated antibody is one that is substantiallyfree of other antibodies with different antigenic specificities than theisolated antibody. For example, in a particular embodiment, apreparation of an antibody described herein is substantially free ofcellular material and/or chemical precursors. The language“substantially free of cellular material” includes preparations of anantibody in which the antibody is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. Thus, anantibody that is substantially free of cellular material includespreparations of antibody having less than about 30%, 20%, 10%, 5%, 2%,1%, 0.5%, or 0.1% (by dry weight) of heterologous protein (also referredto herein as a “contaminating protein”) and/or variants of an antibody,for example, different post-translational modified forms of an antibodyor other different versions of an antibody (e.g., antibody fragments).When the antibody is recombinantly produced, it is also generallysubstantially free of culture medium, i.e., culture medium representsless than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of theprotein preparation. When the antibody is produced by chemicalsynthesis, it is generally substantially free of chemical precursors orother chemicals, i.e., it is separated from chemical precursors or otherchemicals which are involved in the synthesis of the protein.Accordingly, such preparations of the antibody have less than about 30%,20%, 10%, or 5% (by dry weight) of chemical precursors or compoundsother than the antibody of interest. In a specific embodiment,antibodies described herein are isolated or purified.

Antibodies or fragments thereof that specifically bind to CD137 (e.g.,human CD137 or cynomolgus CD137) can be produced by any method known inthe art for the synthesis of antibodies, for example, by chemicalsynthesis or by recombinant expression techniques. The methods describedherein employ, unless otherwise indicated, conventional techniques inmolecular biology, microbiology, genetic analysis, recombinant DNA,organic chemistry, biochemistry, PCR, oligonucleotide synthesis andmodification, nucleic acid hybridization, and related fields within theskill of the art. These techniques are described, for example, in thereferences cited herein and are fully explained in the literature. See,e.g., Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989),Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Ausubel F M et al., Current Protocols in MolecularBiology, John Wiley & Sons (1987 and annual updates); Current Protocolsin Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.)(1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press;Eckstein (ed.) (1991) Oligonucleotides and Analogues: A PracticalApproach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: ALaboratory Manual, Cold Spring Harbor Laboratory Press, all of which areherein incorporated by reference in their entireties.

In a specific embodiment, an antibody described herein is an antibody(e.g., recombinant antibody) prepared, expressed, created or isolated byany means that involves creation, e.g., via synthesis, geneticengineering of DNA sequences. In certain embodiments, such an antibodycomprises sequences (e.g., DNA sequences or amino acid sequences) thatdo not naturally exist within the antibody germline repertoire of ananimal or mammal (e.g., human) in vivo.

In one aspect, provided herein is a method of making an antibody whichspecifically binds to CD137 (e.g., human CD137 or cynomolgus CD137)comprising culturing a cell or host cell described herein. In oneembodiment, the method is performed in vitro. In a certain aspect,provided herein is a method of making an antibody which specificallybinds to CD137 (e.g., human CD137 or cynomolgus CD137) comprisingexpressing (e.g., recombinantly expressing) the antibody using a cell orhost cell described herein (e.g., a cell or a host cell comprisingpolynucleotides encoding an antibody described herein). In a particularembodiment, the cell is an isolated cell. In a particular embodiment,the exogenous polynucleotides have been introduced into the cell. In aparticular embodiment, the method further comprises the step ofpurifying the antibody obtained from the cell or host cell.

Methods for producing polyclonal antibodies are known in the art (see,for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002)5th Ed., Ausubel F M et al., eds., John Wiley and Sons, New York, whichis herein incorporated by reference in its entirety).

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow E & Lane D,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988); Hammerling G J et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981), each of which isherein incorporated by reference in its entirety. The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. For example, monoclonal antibodies can be producedrecombinantly from host cells exogenously expressing an antibodydescribed herein or a fragment thereof, for example, light chain and/orheavy chain of such antibody.

In specific embodiments, a “monoclonal antibody,” as used herein, is anantibody produced by a single cell (e.g., hybridoma or host cellproducing a recombinant antibody), wherein the antibody specificallybinds to CD137 (e.g., human CD137 or cynomolgus CD137) as determined,e.g., by ELISA or other antigen-binding or competitive binding assayknown in the art or in the examples provided herein. In particularembodiments, a monoclonal antibody can be a chimeric antibody or ahumanized antibody. In certain embodiments, a monoclonal antibody is amonovalent antibody or multivalent (e.g., bivalent) antibody. Inparticular embodiments, a monoclonal antibody is a monospecific ormultispecific antibody (e.g., bispecific antibody). Monoclonalantibodies described herein can, for example, be made by the hybridomamethod as described in Kohler G & Milstein C (1975) Nature 256: 495,which is herein incorporated by reference in its entirety, or can, e.g.,be isolated from phage libraries using the techniques as describedherein, for example. Other methods for the preparation of clonal celllines and of monoclonal antibodies expressed thereby are well known inthe art (see, for example, Chapter 11 in: Short Protocols in MolecularBiology, (2002) 5th Ed., Ausubel F M et al., supra).

As used herein, an antibody binds to an antigen multivalently (e.g.,bivalently) when the antibody comprises at least two (e.g., two or more)monovalent binding domains, each monovalent binding domain capable ofbinding to an epitope on the antigen. Each monovalent binding domain canbind to the same or different epitopes on the antigen.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. For example,in the hybridoma method, a mouse or other appropriate host animal, suchas a sheep, goat, rabbit, rat, hamster or macaque monkey, is immunizedto elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the protein (e.g., CD137(e.g., human CD137 or cynomolgus CD137)) used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding J W (Ed),Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986), herein incorporated by reference in its entirety).Additionally, a RIMMS (repetitive immunization multiple sites) techniquecan be used to immunize an animal (Kilpatrick K E et al., (1997)Hybridoma 16:381-9, herein incorporated by reference in its entirety).

In certain embodiments, mice (or other animals, such as rats, monkeys,donkeys, pigs, sheep, hamster, or dogs) can be immunized with an antigen(e.g., CD137 (e.g., human CD137 or cynomolgus CD137)) and once an immuneresponse is detected, e.g., antibodies specific for the antigen aredetected in the mouse serum, the mouse spleen is harvested andsplenocytes isolated. The splenocytes are then fused by well-knowntechniques to any suitable myeloma cells, for example, cells from cellline SP20 available from the American Type Culture Collection (ATCC®)(Manassas, Va.), to form hybridomas. Hybridomas are selected and clonedby limited dilution. In certain embodiments, lymph nodes of theimmunized mice are harvested and fused with NS0 myeloma cells.

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Specific embodiments employ myeloma cells that fuse efficiently, supportstable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these myeloma cell lines are murine myeloma lines, such asthe NS0 cell line or those derived from MOPC-21 and MPC-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,Calif., USA, and SP-2 or X63-Ag8.653 cells available from the AmericanType Culture Collection, Rockville, Md., USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor D (1984) J Immunol133: 3001-5; Brodeur et al., Monoclonal Antibody Production Techniquesand Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), eachof which is herein incorporated by reference in its entirety).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against CD137 (e.g., humanCD137 or cynomolgus CD137). The binding specificity of monoclonalantibodies produced by hybridoma cells is determined by methods known inthe art, for example, immunoprecipitation or by an in vitro bindingassay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbentassay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding J W (Ed), Monoclonal Antibodies: Principles and Practice,supra). Suitable culture media for this purpose include, for example,D-MEM or RPMI 1640 medium. In addition, the hybridoma cells may be grownin vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

Antibodies described herein include, e.g., antibody fragments whichrecognize a specific CD137 (e.g., human CD137 or cynomolgus CD137), andwhich can be generated by any technique known to those of skill in theart. For example, Fab and F(ab′)₂ fragments described herein can beproduced by proteolytic cleavage of immunoglobulin molecules, usingenzymes such as papain (to produce Fab fragments) or pepsin (to produceF(ab′)₂ fragments). A Fab fragment corresponds to one of the twoidentical arms of an antibody molecule and contains the complete lightchain paired with the VH and CH1 domains of the heavy chain. A F(ab′)₂fragment contains the two antigen-binding arms of an antibody moleculelinked by disulfide bonds in the hinge region.

Further, the antibodies described herein can also be generated usingvarious phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.In particular, DNA sequences encoding VH and VL domains are amplifiedfrom animal cDNA libraries (e.g., human or murine cDNA libraries ofaffected tissues). The DNA encoding the VH and VL domains are recombinedtogether with a scFv linker by PCR and cloned into a phagemid vector.The vector is electroporated in E. coli and the E. coli is infected withhelper phage. Phage used in these methods are typically filamentousphage including fd and M13, and the VH and VL domains are usuallyrecombinantly fused to either the phage gene III or gene VIII. Phageexpressing an antigen binding domain that binds to a particular antigencan be selected or identified with antigen, e.g., using labeled antigenor antigen bound or captured to a solid surface or bead. Examples ofphage display methods that can be used to make the antibodies describedherein include those disclosed in Brinkman U et al., (1995) J ImmunolMethods 182: 41-50; Ames R S et al., (1995) J Immunol Methods 184:177-186; Kettleborough C A et al., (1994) Eur J Immunol 24: 952-958;Persic L et al., (1997) Gene 187: 9-18; Burton D R & Barbas C F (1994)Advan Immunol 57: 191-280; PCT Application No. PCT/GB91/001134;International Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, and WO 97/13844; andU.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908,5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225,5,658,727, 5,733,743 and 5,969,108, all of which are herein incorporatedby reference in their entireties.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described below. Techniques to recombinantly produceantibody fragments such as Fab, Fab′ and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication No. WO 92/22324; Mullinax R L et al., (1992) BioTechniques12(6): 864-9; Sawai H et al., (1995) Am J Reprod Immunol 34: 26-34; andBetter M et al., (1988) Science 240: 1041-1043, all of which are hereinincorporated by reference in their entireties.

In certain embodiments, to generate whole antibodies, PCR primersincluding VH or VL nucleotide sequences, a restriction site, and aflanking sequence to protect the restriction site can be used to amplifythe VH or VL sequences from a template, e.g., scFv clones. Utilizingcloning techniques known to those of skill in the art, the PCR amplifiedVH domains can be cloned into vectors expressing a VH constant region,and the PCR amplified VL domains can be cloned into vectors expressing aVL constant region, e.g., human kappa or lambda constant regions. The VHand VL domains can also be cloned into one vector expressing thenecessary constant regions. The heavy chain conversion vectors and lightchain conversion vectors are then co-transfected into cell lines togenerate stable or transient cell lines that express full-lengthantibodies, e.g., IgG, using techniques known to those of skill in theart.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules. Forexample, a chimeric antibody can contain a variable region of a mouse orrat monoclonal antibody fused to a constant region of a human antibody.Methods for producing chimeric antibodies are known in the art. See,e.g., Morrison S L (1985) Science 229: 1202-7; Oi V T & Morrison S L(1986) BioTechniques 4: 214-221; Gillies S D et al., (1989) J ImmunolMethods 125: 191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567,4,816,397, and 6,331,415, all of which are herein incorporated byreference in their entireties.

A humanized antibody is capable of binding to a predetermined antigenand which comprises a framework region having substantially the aminoacid sequence of a human immunoglobulin and CDRs having substantiallythe amino acid sequence of a non-human immunoglobulin (e.g., a murineimmunoglobulin). In particular embodiments, a humanized antibody alsocomprises at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. The antibody also can includethe CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Ahumanized antibody can be selected from any class of immunoglobulins,including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG₁,IgG₂, IgG₃ and IgG₄. Humanized antibodies can be produced using avariety of techniques known in the art, including but not limited to,CDR-grafting (European Patent No. EP 239400; International PublicationNo. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and5,585,089), veneering or resurfacing (European Patent Nos. EP 592106 andEP 519596; Padlan E A (1991) Mol Immunol 28(4/5): 489-498; Studnicka G Met al., (1994) Prot Engineering 7(6): 805-814; and Roguska M A et al.,(1994) PNAS 91: 969-973), chain shuffling (U.S. Pat. No. 5,565,332), andtechniques disclosed in, e.g., U.S. Pat. Nos. 6,407,213, 5,766,886,International Publication No. WO 93/17105; Tan P et al., (2002) JImmunol 169: 1119-25; Caldas C et al., (2000) Protein Eng. 13(5):353-60; Morea V et al., (2000) Methods 20(3): 267-79; Baca M et al.,(1997) J Biol Chem 272(16): 10678-84; Roguska M A et al., (1996) ProteinEng 9(10): 895 904; Couto J R et al., (1995) Cancer Res. 55 (23 Supp):5973s-5977s; Couto J R et al., (1995) Cancer Res 55(8): 1717-22; SandhuJ S (1994) Gene 150(2): 409-10 and Pedersen J T et al., (1994) J MolBiol 235(3): 959-73, all of which are herein incorporated by referencein their entireties. See also U.S. Application Publication No. US2005/0042664 A1 (Feb. 24, 2005), which is herein incorporated byreference in its entirety.

Methods for making multispecific (e.g., bispecific antibodies) have beendescribed, see, for example, U.S. Pat. Nos. 7,951,917; 7,183,076;8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992 and 8,586,713, allof which are herein incorporated by reference in their entireties.

Single domain antibodies, for example, antibodies lacking the lightchains, can be produced by methods well known in the art. See RiechmannL & Muyldermans S (1999) J Immunol 231: 25-38; Nuttall S D et al.,(2000) Curr Pharm Biotechnol 1(3): 253-263; Muyldermans S, (2001) JBiotechnol 74(4): 277-302; U.S. Pat. No. 6,005,079; and InternationalPublication Nos. WO 94/04678, WO 94/25591 and WO 01/44301, all of whichare herein incorporated by reference in their entireties.

Further, antibodies that specifically bind to a CD137 (e.g., human CD137or cynomolgus CD137) antigen can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” an antigen using techniques wellknown to those skilled in the art. See, e.g., Greenspan N S & Bona C A(1989) FASEB J 7(5): 437-444; and Nissinoff A (1991) J Immunol 147(8):2429-2438, each of which is herein incorporated by reference in itsentirety.

In particular embodiments, an antibody described herein, which binds tothe same epitope of CD137 (e.g., human CD137 or cynomolgus CD137) as ananti-CD137 (e.g., human CD137 or cynomolgus CD137) antibody describedherein, is a human antibody. In particular embodiments, an antibodydescribed herein, which competitively blocks (e.g., in a dose-dependentmanner) any one of the antibodies described herein, from binding toCD137 (e.g., human CD137 or cynomolgus CD137), is a human antibody.Human antibodies can be produced using any method known in the art. Forexample, transgenic mice which are incapable of expressing functionalendogenous immunoglobulins, but which can express human immunoglobulingenes, can be used. In particular, the human heavy and light chainimmunoglobulin gene complexes can be introduced randomly or byhomologous recombination into mouse embryonic stem cells. Alternatively,the human variable region, constant region, and diversity region can beintroduced into mouse embryonic stem cells in addition to the humanheavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes can be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of theJ_(H) region prevents endogenous antibody production. The modifiedembryonic stem cells are expanded and microinjected into blastocysts toproduce chimeric mice. The chimeric mice are then bred to producehomozygous offspring which express human antibodies. The transgenic miceare immunized in the normal fashion with a selected antigen, e.g., allor a portion of an antigen (e.g., CD137 (e.g., human CD137 or cynomolgusCD137)). Monoclonal antibodies directed against the antigen can beobtained from the immunized, transgenic mice using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg N & Huszar D (1995) Int RevImmunol 13:65-93, herein incorporated by reference in its entirety. Fora detailed discussion of this technology for producing human antibodiesand human monoclonal antibodies and protocols for producing suchantibodies, see, e.g., International Publication Nos. WO 98/24893, WO96/34096 and WO 96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126,5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318 and 5,939,598, allof which are herein incorporated by reference in their entireties.Examples of mice capable of producing human antibodies include theXenomouse™ (Abgenix, Inc.; U.S. Pat. Nos. 6,075,181 and 6,150,184), theHuAb-Mouse™ (Mederex, Inc./Gen Pharm; U.S. Pat. Nos. 5,545,806 and5,569,825), the Trans Chromo Mouse™ (Kirin) and the KM Mouse™(Medarex/Kirin), all of which are herein incorporated by reference intheir entireties.

Human antibodies that specifically bind to CD137 (e.g., human CD137 orcynomolgus CD137) can be made by a variety of methods known in the artincluding the phage display methods described above using antibodylibraries derived from human immunoglobulin sequences. See also U.S.Pat. Nos. 4,444,887, 4,716,111, and 5,885,793; and InternationalPublication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741, all of which are hereinincorporated by reference in their entireties.

In certain embodiments, human antibodies can be produced usingmouse-human hybridomas. For example, human peripheral blood lymphocytestransformed with Epstein-Barr virus (EBV) can be fused with mousemyeloma cells to produce mouse-human hybridomas secreting humanmonoclonal antibodies, and these mouse-human hybridomas can be screenedto determine ones which secrete human monoclonal antibodies thatspecifically bind to a target antigen (e.g., CD137 (e.g., human CD137 orcynomolgus CD137)). Such methods are known and are described in the art,see, e.g., Shinmoto H et al., (2004) Cytotechnology 46: 19-23; NaganawaY et al., (2005) Human Antibodies 14: 27-31, each of which is hereinincorporated by reference in its entirety.

5.6 Kits

Also provided are kits comprising one or more antibodies describedherein, or pharmaceutical compositions or conjugates thereof. In aspecific embodiment, provided herein is a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions described herein, such asone or more antibodies provided herein. In certain embodiments, the kitscontain a pharmaceutical composition described herein and anyprophylactic or therapeutic agent, such as those described herein. Incertain embodiments, the kits may contain a T cell mitogen, such as,e.g., phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA),or a TCR complex stimulating antibody, such as an anti-CD3 antibody andanti-CD28 antibody. Optionally associated with such container(s) can bea notice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

Also provided, are kits that can be used in the above methods. In oneembodiment, a kit comprises an antibody described herein, preferably apurified antibody, in one or more containers. In a specific embodiment,kits described herein contain a substantially isolated CD137 (e.g.,human CD137 or cynomolgus CD137) antigen as a control. In anotherspecific embodiment, the kits described herein further comprise acontrol antibody which does not react with a CD137 (e.g., human CD137 orcynomolgus CD137) antigen. In another specific embodiment, kitsdescribed herein contain one or more elements for detecting the bindingof an antibody to a CD137 (e.g., human CD137 or cynomolgus CD137)antigen (e.g., the antibody can be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody can be conjugated to a detectablesubstrate). In specific embodiments, a kit provided herein can include arecombinantly produced or chemically synthesized CD137 (e.g., humanCD137 or cynomolgus CD137) antigen. The CD137 (e.g., human CD137 orcynomolgus CD137) antigen provided in the kit can also be attached to asolid support. In a more specific embodiment, the detecting means of theabove described kit includes a solid support to which a CD137 (e.g.,human CD137 or cynomolgus CD137) antigen is attached. Such a kit canalso include a non-attached reporter-labeled anti-human antibody oranti-mouse/rat antibody. In this embodiment, binding of the antibody tothe CD137 (e.g., human CD137 or cynomolgus CD137) antigen can bedetected by binding of the said reporter-labeled antibody. In oneembodiment, the present invention relates to the use of a kit of thepresent invention for in vitro assaying and/or detecting CD137 antigen(e.g., human CD137 or cynomolgus CD137) in a biological sample.

6. EXAMPLES

The examples in this Section (i.e., Section 6) are offered by way ofillustration, and not by way of limitation.

6.1 Example 1: Characterization of an Anti-CD137 Antibody

This example describes the characterization of antibodies that bind tohuman CD137. In particular, the BA001 antibody, which specifically bindsto and stimulates the function of human CD137, was characterized. Thesequence information of the variable regions of BA001 is provided inTable 1.

6.1.1 Anti-Human CD137 Antibody Binds to Cells Expressing CD137.

The capacity of the human anti-CD137 IgG1 antibody BA001 to bind tocells expressing human CD137 or cynomolgus monkey CD137 was tested in avariety of cell types, as shown in FIGS. 1A and 1B.

Engineered Jurkat Cells

In one example, Jurkat cells were engineered to constitutively expresseither human CD137 or cynomolgus CD137 and used to analyze the bindingof antibody BA001. Briefly, transfected Jurkat cells were plated in a96-well round bottom plate at 5×10⁴ cells/well, and incubated withserial dilutions of antibody (i.e., BA001 or isotype control, at theindicated concentrations) for 25 minutes at 4° C. (left panels of FIGS.1A and 1B). The cells were washed twice and incubated with an anti-humanlambda-PE secondary antibody (Life Technologies, Cat #MH10614). Thecells were washed and suspended in 80 μl of 2% paraformaldehyde(Electron Microscopy Sciences) prepared in PBS. Data were collected withBD FACS Canto and analyzed using BD FACSDiva software.

As shown in FIGS. 1A and 1B (left panels), the BA001 antibody bound toJurkat cells expressing either human CD137 or cynomolgus CD137.

Activated CFM/C1 T Cells

In a second example, the capacity of BA001 to bind to activated humanCEM/C1 T cells expressing endogenous human CD137 was tested. Briefly,CEM/C1 cells were stimulated by incubation with 10 ng/ml Phorbol12-myristate 13-acetate (PMA) and 1 μg/ml ionomycin at 37° C. for 18hours. The stimulated cells were plated in a 96-well round bottom plateat 1×10⁵ cells/well and incubated with serial dilutions of antibody(i.e., BA001 or isotype control, at concentrations shown in the middlepanel of FIG. 1A) for 25 minutes at 4° C. The cells were washed twiceand incubated with an anti-human lambda-PE secondary antibody (LifeTechnologies, Cat #MH10614). The cells were washed and suspended in 80μl of 2% paraformaldehyde (Electron Microscopy Sciences) prepared inPBS. Data were collected with BD FACS Canto and analyzed using BDFACSDiva software.

As shown in the middle panel of FIG. 1A, the BA001 antibody bound toactivated CEM/C1 cells expressing endogenous CD137.

Activated Primary CD8+ T Cells

In a third example, the capacity of BA001 to bind to activated human orcynomolgus CD8+ T cells was tested. Briefly, human or cynomolgus PBMCswere stimulated by incubation with 10 ng/ml PMA and 1 μg/ml ionomycin at37° C. for 18 hours. The stimulated cells were plated in a 96-well roundbottom plate at 1×10⁵ cells/well and incubated with serial dilutions ofantibody (i.e., BA001 or isotype control, at concentrations shown in theright panels of FIGS. 1A and 1B) and anti-human CD8-APC (Biolegend, Cat#311049) for 25 minutes at 4° C. The cells were washed twice andincubated with an F(ab′)₂ goat anti-human IgG-PE secondary antibody(Jackson ImmunoResearch, Cat #109-116-098). The cells were washed andsuspended in 80 μl of 2% paraformaldehyde (Electron Microscopy Sciences)prepared in PBS. Data were collected with BD FACS Canto, and thenanalyzed (gated on CD8+ T cells) using Flowjo V10.

As shown in the right panels of FIGS. 1A-1B, the BA001 antibody bound toactivated human or cynomolgus CD8+ T cells expressing endogenous CD137.

6.1.2 Anti-CD137 Antibody does not Block CD137L Binding to CD137.

Binding of CD137L to CD137/BA001-F(Ab)₂ Complex

Surface plasmon resonance was used to evaluate the capacity of CD137L tobind to CD137 complexed to the F(ab′)₂ fragment of BA001(BA001-F(ab′)₂). BA001-F(ab′)₂ was generated using the FragIT™ kit,Genovis (Cat #A2-FR2-100). All interactions were analyzed at 25° C.using a BIAcore® T200 (GE Healthcare) and 1×HBS-P+ (GE Healthcare,BR-1006-71) as running buffer.

In one example, BA001-F(ab′)₂ was immobilized on a chip and then boundto CD137, after which CD137L was permitted to bind to theCD137/BA001-F(ab′)₂ complex. First, an anti-human Fab capture antibody(GE Healthcare, Fab Capture Kit, 28-9583-25) was immobilized on flowcell 2 of a CM5 series S sensor chip (GE Healthcare, 29-1496-03).BA001-F(ab′)₂ was then diluted in 6.75 μg/ml in running buffer andimmobilized to flow cell 1 at 10 μl/min for 120 seconds. As a controlfor measuring non-specific interaction of CD137 or CD137L, flow cell 1of the chip was bound solely with the anti-human Fab capture antibody.After capture of BA001-F(ab′)₂, 100 nM of CD137 (Acro Biosystem,41B-H5227) was run at 30 μl/min for 90 seconds over both flow cells ofthe chip, followed by 400 seconds of dissociation. 200 nM of CD137L (R&DSystems, #2295-4L-025) was then run at 30 μl/min for 90 seconds overboth flow cells, followed by a dissociation time of 400 seconds.

The response obtained for flow cell 2 minus the response obtained forflow cell 1 is shown in FIG. 2A. When CD137 was run over flow cell 2, anincrease in the response signal was detected, showing the binding ofCD137 to BA001-F(ab′)₂. CD137 was seen to dissociate very slowly fromBA001-F(ab′)₂. CD137L was then run over flow cell 2 and an increase ofthe signal response was observed, showing the binding of CD137L to theCD137/BA001-F(ab′)₂ complex. These results show that binding ofBA001-F(ab′)₂ to CD137 does not block the binding of CD137L to CD137.

In another example, an excess of CD137 (110 nM) was pre-mixed withBA001-F(ab′)₂ (6 μg/ml, 54 nM) to form a CD137/BA001-F(ab′)₂ complex.The complex was then immobilized on flow cell 3 of a CM5 series S sensorchip (GE Healthcare, 29-1496-03) at 10 μl/min for 180 seconds, followedby 60 seconds of dissociation. 60 nM of CD137L was then run at 50 μl/minfor 90 seconds over all the flow cells followed by a dissociation timeof 400 seconds. As a control for measuring non-specific interaction ofCD137 or CD137L, flow cell 1 of the chip was bound solely with theanti-human Fab capture antibody.

The response obtained for flow cell 3 minus the response obtained forflow cell 1 is shown in FIG. 2B. These data demonstrated that althoughBA001-F(ab′)₂ bound to CD137 with high affinity, this interaction doesnot impair the binding of CD137L to CD137.

Similar results to those described in this example were also observedfor BA001 and a Fab fragment derived from BA001 (BA001-Fab; data notshown). Thus, BA001 is a non-ligand blocking anti-CD137 antibody.

BA001 Did not Block Cell Surface CD137L Binding to Cell Surface CD137

To determine whether BA001 can block binding between CD137L and CD137expressed on the surface of cells (i.e., in a more physiologicalsetting), a cell conjugation assay was performed using the methodologydescribed in Xiao et al. (JEM211(5):943-959, 2014; incorporated hereinby reference in its entirety). Briefly, one set of Jurkat cells wastransfected with human CD137 (Jurkat-CD137) and another set of Jurkatcells was transfected with human CD137L (Jurkat-CD137L).CD137-expressing Jurkat cells were stained with the red dye PKH26 (SigmaCat #PKH26GL-1KT) and CD137L-expressing Jurkat cells were stained withthe green dye PKH67 (Sigma Cat #PKH67GL-1KT). Red dye-labeledJurkat-CD137 cells (1×10⁵/well) were incubated with 50 μg/ml of BA001,reference anti-CD137 antibody #1, reference anti-CD137 antibody #2, orisotype control in a round-bottom 96-well plate for 30 min at roomtemperature. Then green dye-labeled Jurkat-CD137L cells (1×10⁵/well)were added and incubated for 45 min at 37° C. Cell to cellbinding/conjugate formation was analyzed by flow cytometry using BD FACSCanto and BD FACSDiva software. The PE channel was used for the red dyeand the FITC channel was used for the green dye. As such, bindingbetween a CD137-expressing cell and a CD137L-expressing cell wouldresult in a double-positive signal (i.e., red+green) that exhibitsincreased detected cell size. This effect would be reduced or abolishedby a ligand blocking anti-CD137 antibody.

FIG. 3A shows that BA001 and reference anti-CD137 antibody #2 did notblock CD137L on cells from binding to CD137 on cells. By contrast,reference anti-CD137 antibody #1 blocked ligand binding.

In a similar setting, CD137L-expressing and CD137-expressing cells werestained with the PKH26 red fluorescent cell linker or PKH67 greenfluorescent cell linker, respectively, and suspended at a concentrationof 4×10⁶ cells/mL in Hanks' balanced salt solution (HBSS). A 3-foldserial dilution of BA001, reference anti-CD137 antibody #1, referenceanti-CD137 antibody #2, or the respective isotype control antibody wereprepared in HBSS at a 3× working concentration. In a U-bottom 96-wellplate, 25 uL of Jurkat-CD137 cells were incubated at RT with 25 uL of ananti-CD137 antibody for 30 min, and CD137L-expressing cells were added.Alternatively, 25 uL of Jurkat-CD137 cells were incubated at RT with 25uL of the CD137L-expressing cells for 30 min, and an anti-CD137 antibodywas added. The plate was incubated for 45 minutes at 37 C and 5% CO2,and conjugates of CD137L-expressing cells and CD137-expressing cellswere identified as PE and FITC double positivity by flow cytometry usingthe BD Fortessa cytometer.

As shown in FIGS. 3B and 3C, BA001 and reference anti-CD137 antibody #2did not affect the conjugation of CD137-expressing cells with theCD137L-expressing cells, when the anti-CD137 antibody was added prior to(FIG. 3B) or after (FIG. 3C) co-culture of the cells. By contrast,reference anti-CD137 antibody #1 inhibited the cell conjugation,indicating that this antibody blocked cell surface CD137L binding tocell surface CD137.

6.2 Example 2: The Agonistic Activity of Anti-CD137 Antibody isCrosslinking- and Ligand-Dependent 6.2.1 Anti-CD137 Antibody InducesNF-κB-Driven Gene Expression Only in the Presence of AntibodyCrosslinking.

To characterize the ability of BA001 to activate CD137 signaling, Jurkatreporter cells were generated that incorporated (i) an NFκB-luciferasereporter construct, and (ii) an expression construct for either human orcynomolgus CD137. As such, activation of CD137 on the surface of thereporter cells induced downstream signaling that drives expression ofluciferase under the control of the NFκB promoter.

It was discovered that the ability of BA001 to activate CD137 wasdependent on BA001 crosslinking. Indeed, uncrosslinked BA001 was unableto stimulate reporter activity in Jurkat cells engineered to expressCD137 and an NF-κB luciferase reporter construct (data not shown). Tocharacterize the crosslinking dependency, BA001, an isotype controlantibody, and reference anti-CD137 antibody #2 were incubated with adose titration of crosslinker (AffiniPure F(ab′)₂ Fragment GoatAnti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch,109-006-098)). The Jurkat reporter cells were seeded at the density of50,000 cells/well, and were incubated with 2 μg/mL BA001, an isotypecontrol antibody, or reference anti-CD137 antibody #2 for 4 hours. TheNF-kB activities were measured by Nano-Glo® Luciferase Assay System(Promega N1120).

As shown in FIG. 4A, BA001 acquired activity in this assay when it wascrosslinked at a crosslinker-to-antibody ratio above 1:1. By contrast,reference anti-CD137 antibody #2 was active without crosslinker, andgradually lost activity with an increasing amount of crosslinker.

It was further assessed whether BA001 was able to agonize CD137 uponantibody clustering in the absence of an artificial antibodycrosslinker. Antibody clustering was induced by CHO cells engineered toexpress FcγRIIIa (CD16). Briefly, the Jurkat reporter cells at thedensity of 50,000 cells/well were co-cultured with a dose titration ofCHO cells engineered to express CD16 or control CHO cells, in thepresence of 2 μg/mL of BA001, an isotype control antibody, or a BA001variant having an N297A mutation in the Fc region. The NF-kB activitieswere measured by Nano-Glo® Luciferase Assay System (Promega N1120) after4 hours of incubation.

As shown in FIG. 4B, BA001 alone did not activate CD137 signaling, andCD16-expressing CHO cells alone had a limited effect. However, thecombination of BA001 and CD16-expressing CHO cells synergisticallyactivated the reporter cells. The N297A mutation abrogated the abilityof BA001 to stimulate CD137 signaling, likely because the N297A Fcvariant was not able to engage CD16 expressed on the CHO cells, and thuswas not able to undergo antibody clustering. This result suggests thatBA001 may be selectively active in a microenvironment whereinCD16-expressing cells (e.g., antigen-presenting cells or NK cells) arepresent.

6.2.2 Anti-CD137 Antibody Enhances T Cell Function with Whole PBMC butnot with Purified T Cells.

BA001 Promoted IL-2 Secretion by Human T Cells in the Presence of CD137L

The agonistic activity of BA001 on primary human PBMCs was assessedfollowing Staphylococcal Enterotoxin A (SEA) stimulation. Briefly,cryopreserved PBMCs were stimulated with 200 ng/ml of the SEAsuperantigen (Toxin Technologies, Cat #AT101red) in the presence ofserial dilutions of an antibody (i.e., BA001, reference anti-CD137antibody #1 or #2, or an isotype control antibody, at the concentrationsshown in FIG. 5) for 5 days at 37° C. IL-2 concentrations in the culturesupernatant were analyzed by AlphaLISA (Perkin Elmer, Cat #AL221F). Eachcondition was tested with five replicates.

As shown in FIG. 5, the anti-CD137 antibody BA001 (IgG1) increased IL-2production in human PBMCs in a dose-dependent manner, at levelscomparable or greater than those of reference anti-CD137 antibodies.

BA001 Did not Promote IL-2 Secretion by Purified Human T Cells in theAbsence of CD137L

The agonistic activity of BA001 on purified, stimulated human T cells inthe absence of antigen-presenting cells expressing CD137L was assessed.Briefly, T cells were purified from cryopreserved PBMCs using MACS Pan TCell Isolation Kit (human) with autoMACS columns following themanufacturer instructions. The purified T cells were plated at 1×10⁶cells/well into 96-well culture plates pre-coated with low endotoxin,azide-free (LEAF) anti-CD3 antibody (Biolegend Cat #300432) at 2 μg/ml.Anti-CD137 antibody (BA001, reference anti-CD137 antibody #1, orreference anti-CD137 antibody #2) or isotype control at 5 μg/ml wascross-linked with F(ab′)₂ fragment goat anti-human IgG (JacksonImmunoResearch, Cat #109-006-098) and then added to the plate. Cellswere incubated at 37° C. for three days. IL-2 concentrations in theculture supernatant were then analyzed by AlphaLISA (Perkin Elmer, Cat#AL221F). Each condition was tested with six replicates.

As shown in FIGS. 6A-6B, BA001 did not promote increased IL-2 secretionby purified T cells, relative to isotype control. By contrast, bothreference anti-CD137 antibodies induced elevated IL-2 secretion by thepurified T cells. FIG. 6C shows that the purified T cells did notexpress detectable levels of CD137L.

Taken together, the data in Section 6.2.2 show that the agonisticactivity of BA001 was elevated in the presence of CD137L (e.g., producedby CD137L-expressing cells), as in whole PBMCs. It is contemplated thatthe agonistic activity of BA001 may require the presence of CD137L underthe conditions used. Thus, these data demonstrated that the ability ofBA001 to activate CD137-expressing cells might be ligand-dependent.

6.2.3 Anti-CD137 Antibody Only Induces NFκB-Driven Gene Expression inthe Presence of CD137L. Ligand Dependency of BA001 in NFκB-LuciferaseReporter Cells

It was shown in section 6.2.1 that BA001 activity was dependent oncrosslinking in the absence of CD137L. The effect of crosslinker in thepresence of CD137L was further assessed. Briefly, 1 μg/mL CD137L(Recombinant Human 4-1BB Ligand/TNFSF9 (His-tag), R&D system,2295-4L-025/CF) was optionally added to the culture system describedabove, and the NF-κB activity was measured similarly.

As shown in FIG. 7A, in the presence of CD137L, BA001 still required thecrosslinker for activity in the reporter assay, and the effects ofCD137L and crosslinking were additive. When the crosslinker-to-antibodyratio was about 1:10 to 1:1, BA001 showed activity only in the presenceof CD137L.

To confirm that the exogenous CD137L was the sole source of CD137L inthis experimental system, the Jurkat reporter cells were analyzed byflow cytometry. Briefly, CD137-expressing and CD137L-expressing Jurkatcells were thawed and cultured in RPMI medium supplemented with 10%fetal bovine serum and 1 μg/mL puromycin for 4 or 24 hours. For analysisby flow cytometry, 3×10⁴ cells were plated in a 96 well U-bottom plate,washed twice with cold phosphate buffer saline supplement with 2% fetalbovine serum, and labeled with an anti-CD137 antibody conjugated withphycoerythrin (PE), an anti-CD137L antibody conjugated withallophycocyanin (APC), and a near-IR live/dead dye. In the stainingcontrol group, the cells were labeled with an irrelevant isotype controlantibody conjugated with PE, an irrelevant isotype control antibodyconjugated with APC, and the live/dead dye.

As shown in FIG. 7B, the CD137-expressing Jurkat reporter cellsexpressed high levels of CD137 but not CD137L. In comparison, theCD137L-expressing Jurkat cells expressed high levels of CD137L but notCD137.

In all the NF-κB activation assays below using Jurkat reporter cells,anti-CD137 antibodies and their isotype control antibodies werecrosslinked at a ratio of 1:2.

In one example, Jurkat NFκB-luciferase reporter cells expressing humanCD137 (50,000 cells/well) were incubated with serial dilutions of BA001or an isotype control, in the presence or absence of soluble humanCD137L (125 ng/ml), for four hours at 37° C. Luciferase expression wasdetected using the Nano-Glo® Luciferase Assay System (Promega Cat#N1120) and an EnVision plate reader. As shown in FIG. 8A, BA001 did notinduce NFκB-luciferase expression in the absence of CD137L. In thepresence of CD137L, BA001 was able to induce NFκB-luciferase expressionin a dose-dependent manner (FIG. 8B).

In another example, Jurkat NFκB-luciferase reporter cells expressingcynomolgus CD137 (50,000 cells/well) were incubated with serialdilutions of BA001 or an isotype control, in the presence or absence ofsoluble human CD137L (150 ng/ml), for four hours at 37° C. Luciferaseexpression was detected using the Nano-Glo® Luciferase Assay System(Promega Cat #N1120) and an EnVision plate reader. As shown in FIG. 8C,BA001 did not induce NFκB-luciferase expression in the absence ofCD137L. In the presence of CD137L, BA001 was able to induceNFκB-luciferase expression in a dose-dependent manner (FIG. 8D).

Thus, these data show that BA001 induces human or cynomolgus CD137signaling through NFκB only in the presence of the corresponding CD137Lunder the conditions used. BA001 cooperated with CD137L to promote CD137signaling

In a further example, Jurkat NFκB-luciferase reporter cells expressinghuman CD137 (50,000 cells/well) were incubated with 2 μg/ml ofanti-CD137 antibody (BA001, reference anti-CD137 antibody #1, orreference anti-CD137 antibody #2) or an isotype control, in the presenceof serial dilutions of soluble human CD137L (0-1000 ng/ml, as shown inFIGS. 9A-9C), for four hours at 37° C. In one set of samples, theanti-CD137 antibody was added before the CD137L (FIG. 9A). In a secondset of samples, the anti-CD137 antibody and CD137L were addedsimultaneously (FIG. 9B). In a third set of samples, the CD137L wasadded before the anti-CD137 antibody (FIG. 9C). Luciferase expressionwas detected using the Nano-Glo® Luciferase Assay System (Promega Cat#N1120) and an EnVision plate reader.

As shown in FIGS. 9A-9C, CD137L induced NFκB-luciferase expression in adose-dependent manner. BA001 induced NFκB-luciferase expression in aligand-dependent manner, and substantially increased reporter expressionbeyond that detected for isotype control at higher ligandconcentrations. This effect was observed regardless of the order inwhich antibody and ligand were added (FIGS. 9A-9C, left panels). Bycontrast, the ligand-blocking reference anti-CD137 antibody #1 droveapproximately the same level of reporter expression regardless of theconcentration of CD137L present (e.g., in the absence of CD137L) andregardless of the order in which antibody and ligand were added (FIGS.9A-9C, middle panels). The partially/non-ligand blocking referenceanti-CD137 antibody #2 also drove similar levels of reporter expressionregardless of the concentration of CD137L present (e.g., in the absenceof CD137L) when antibody was added before ligand (FIG. 9A, right panel),but showed substantial reduction of reporter expression at higher CD137Lconcentrations when antibody and ligand were added together (FIG. 9B,right panel) or when ligand was added before antibody (FIG. 9C, rightpanel).

6.3 Characterization of Anti-CD137 Antibody with Different Fc Regions

This example analyzes the impact of Fc/Fc receptor interaction on thefunctional activity of the anti-CD137 antibody BA001. In particular, theVH regions of BA001 were expressed with various Fc backbones, includingIgG2 and IgG4, as well as IgG1 backbones in which the Fc regioncomprises the N297A or S267E/L328F (SELF) mutations, numbered accordingto the EU numbering system, and an IgG2 backbone in which the Fc regioncomprises the N297A mutation. As is known in the art, the IgG1 N297A andIgG2 N297A variants carry an Fc silent mutation that abolishes theengagement of FcγRs, and thus blocks ADCC/ADCP potential orcross-linking of antibodies via FcγRs. The IgG1 SELF Fc variant exhibitsreduced FcγRIIIa binding and enhanced FcγRIIb binding, thus reducingADCC/ADCP potential but enhancing cross-linking of antibodies viaFcγRIIb. In some instances, the N-terminal residue of the heavy chainsequences of anti-CD137 antibodies described herein is glutamine. Insome instances, the N-terminal residue of the heavy chain sequences ofanti-CD137 antibodies described herein is pyroglutamate (e.g., due topost-translational processing).

The antibody BA001 (IgG1) comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 9 and a light chain comprising the aminoacid sequence of SEQ ID NO: 21. The antibody BA001 IgG1 N297A (i.e., theIgG1 N297A variant of BA001) comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 10 and a light chain comprising theamino acid sequence of SEQ ID NO: 21. The antibody BA001 IgG1 SELF(i.e., the IgG1 S267E/L328F variant of BA001) comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 11 and a light chaincomprising the amino acid sequence of SEQ ID NO: 21. The antibody BA001IgG2 (i.e., the IgG2 variant of BA001) comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 12 and a light chaincomprising the amino acid sequence of SEQ ID NO: 21. The antibody BA001IgG2 N297A (i.e., the IgG2 N297A variant of BA001) comprises a heavychain comprising the amino acid sequence of SEQ ID NO: 13 and a lightchain comprising the amino acid sequence of SEQ ID NO: 21. The antibodyBA001 IgG4 (i.e., the IgG4 variant of BA001) comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 14 and a light chaincomprising the amino acid sequence of SEQ ID NO: 21. In some instances,an antibody including an IgG4 Fc region (e.g., antibody BA001 IgG4)includes an S228P mutation that may increase heavy chain stability.These Fc variants of BA001 were then tested in functional assays, asdescribed below.

6.3.1 Fc Variant Functionality in Primary Human PBMCs.

The functional activity of the BA001 Fc variants described above onprimary human PBMCs was assessed following SEA stimulation. Briefly,cryopreserved PBMCs were stimulated with 200 ng/ml of the SEAsuperantigen (Toxin Technologies, Cat #AT101red) in the presence ofserial dilutions of a BA001 Fc variant or the corresponding isotypecontrol antibody for 5 days at 37° C. IL-2 concentrations in the culturesupernatant were analyzed by AlphaLISA (Perkin Elmer, Cat #AL221F). Eachcondition was tested with five replicates.

As shown in FIG. 10A, BA001 IgG1 and BA001 IgG1 SELF each induced strongIL-2 expression in SEA-stimulated primary human PBMCs, which may be dueto enhanced antibody cross-linking of these variants. By contrast, theBA001 IgG1 N297A and BA001 IgG2 N297A variants, which do not formantibody cross-linkages, exhibited little or no detectable function.These data indicated that antibody cross-linking enhanced the functionof BA001 in agonizing CD137 on primary human T cells. BA001 IgG2 andBA001 IgG4 also induced a moderate level of IL-2 expression.

FIG. 10B shows the dose-dependent activity of BA001 Fc variants,excluding the N297A mutants, in enhancing IL-2 expression bySEA-stimulated primary human T cells. BA001 IgG1 SELF and BA001 IgG1induced the most robust effect, followed by BA001 IgG4 and BA001 IgG2.

6.3.2 Fc Variants Maintain Ligand Dependency

The ligand dependency of the BA001 Fc variants was examined using theNFκB-luciferase reporter system described in Section 2.2. Briefly,Jurkat NFκB-luciferase reporter cells expressing human CD137 (50,000cells/well) were incubated with serial dilutions of a BA001 Fc variantor the corresponding isotype control crosslinked by the method describedin Section 6.2.3, in the presence or absence of soluble human CD137L(125 ng/ml), for four hours at 37° C. Luciferase expression was detectedusing the Nano-Glo® Luciferase Assay System (Promega Cat #N1120) and anEnVision plate reader.

As shown in FIG. 11, all of the BA001 Fc variants exhibitedligand-dependent CD137 agonism. In the absence of CD137L, no reporteractivity was detected for any of the Fc variants tested (FIG. 11, leftcolumn), whereas in the presence of CD137L, the BA001 Fc variants allinduced reporter expression in a dose-dependent manner (FIG. 11, rightcolumn). That the cross-linking deficient Fc variants were able toagonize CD137 in this context is likely due to the extremely high levelof CD137 expressed by the reporter cells, in comparison to CD137expression levels in primary T cells.

6.4 Combination Therapies

6.4.1 Combination with Anti-PD-1 Antibody

The anti-CD137 antibody BA001 was further assessed for its ability tostimulate cytokine production by activated T cells, alone or incombination with an anti-PD-1 antibody or an anti-OX40 antibody. In oneexample, cryopreserved primary human PBMCs were stimulated with SEA, asdescribed above, in the presence of either BA001 (5 μg/ml) and isotypecontrol (10 μg/ml), an anti-PD-1 antibody (10 μg/ml) and isotype control(5 μg/ml), a combination of BA001 (5 μg/ml) and the anti-PD-1 antibody(10 μg/ml), or isotype control alone (15 μg/ml). In another example,cryopreserved primary human PBMCs were stimulated with SEA, as describedabove, in the presence of either BA001 (5 μg/ml) and isotype control (10μg/ml), an anti-OX40 antibody (10 μg/ml) and isotype control (5 μg/ml),a combination of BA001 (5 μg/ml) and the anti-OX40 antibody (10 μg/ml),or isotype control alone (15 μg/ml). IL-2 concentrations in the culturesupernatant were analyzed by AlphaLISA (Perkin Elmer, Cat #AL221F). Eachcondition was tested with six replicates.

As shown in FIG. 12A, the combination of BA001 and the anti-PD-1antibody resulted in greater IL-2 secretion than either antibody alone.Similarly, as shown in FIG. 12B, the combination of BA001 and theanti-OX40 antibody induced greater IL-2 secretion than either antibodyalone.

6.5 Epitope Mapping

The interaction of human CD137 with the Fab fragment of BA001(BA001-Fab) or the F(ab′)₂ fragment of BA001 (BA001-F(ab′)₂) was studiedby HDX mass spectrometry. These data were used to identify the epitoperegions bound by BA001-Fab and BA001-F(ab′)₂ on the extracellular domainof human CD137.

6.5.1 Epitope Mapping of Anti-CD137 Antibody by Hydrogen-DeuteriumExchange (HDX)

The interaction of CD137 with anti-human CD137 F(ab′)₂ and anti-humanCD137 Fab were evaluated using the methods below.

(A) CD137 Interaction with Anti-Human CD137 Flab)₂

20 μL human CD137 (5.48 μg) or 20 μL human CD137 and BA001-F(ab′)₂mixture (5.48 μg: 22.36 μg) was incubated with 105 μL deuterium oxidelabeling buffer (50 mM sodium phosphate, 100 mM sodium chloride at pD7.4) for 0 sec, 60 sec, 300 sec, 1800 sec, 7200 sec and 14400 sec at 20°C. Hydrogen/deuterium exchange was quenched by adding 125 μL of 4 Mguanidine hydrochloride, 0.85 M TCEP buffer (final pH is 2.5) andincubating the mixture for 5 min at 20° C. Subsequently, the quenchedsamples were subjected to on column pepsin/protease XIII digestion andLC-MS analysis, as described below. The mass spectra were recorded in MSonly mode.

(B) CD137 Interaction with Anti-Human CD137 Fab

15 μL human CD137 (5.0 μg) or 15 μL human CD137 and BA001-Fab mixture(5.0 human CD137+15.0 μg Fab) was incubated with 110 μL deuterium oxidelabeling buffer (50 mM sodium phosphate, 100 mM sodium chloride at pD7.4) for 0 sec, 60 sec, 300 sec, and 1800 sec at 25° C.Hydrogen/deuterium exchange was quenched by adding 125 μL of 4 Mguanidine hydrochloride, 0.85 M TCEP buffer (final pH is 2.5) andincubating the mixture for 3 min at 25° C. Subsequently, the quenchedsamples were subjected to on column pepsin/protease XIII digestion andLC-MS analysis, as described below. The mass spectra were recorded in MSonly mode.

HDX Data Analysis

Raw MS data was processed using HDX WorkBench software for the analysisof H/D exchange MS data. The deuterium levels were calculated using theaverage mass difference between the deuterated peptide and its nativeform (to). For the calculation of deuterium incorporation, the massspectra for a given peptide were combined across the extracted ionchromatogram peak and the weighted average m/z was calculated. The massincrease from the mass of the native peptide (0 minute) to the weightedaveraged mass corresponds to the level of deuterium incorporation.

Pepsin/Protease XIII Digestion and LC-MS

His-tagged human CD137 (AcroBiosystems Inc.) was fragmented intopeptides for use in HDX by pepsin/protease XII digestion. 5.48 μg ofhuman CD137 in 125 μL control buffer (50 mM phosphate, 100 mM sodiumchloride at pH 7.4) was denatured by adding 125 of 4 M guanidinehydrochloride, 0.85 M TCEP buffer (final pH is 2.5) and incubating themixture for 5 min at 20° C. The mixture was subjected to on-columnpepsin/protease XIII digestion using an in-house packed pepsin/proteaseXIII (w/w, 1:1) column and the resultant peptides were analyzed using anUPLC-MS system comprised of a Waters Acquity UPLC coupled to a QExactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo). Peptideidentification was performed by searching MS/MS data against the humanCD137 sequence with Mascot. The mass tolerance for the precursor andproduct ions was 10 ppm and 0.05 Da, respectively.

Epitope Binding of Anti-Human CD137 Flab)₂

Most of the CD137 peptides displayed identical or similar deuteriumlevels with and without BA001-F(ab′)₂ present. Several peptide segments,however, were found to have significantly decreased deuteriumincorporation upon F(ab′)₂ binding. All the residues in this paragraphare numbered according to SEQ ID NO: 25. One region consisting ofresidues 125-155 (FNDQKRGICRPWTNCSLDGKSVLVNGTKERD, SEQ ID NO: 27),experienced strong deuterium protection when human CD137 was bound toBA001-F(ab′)₂. Thus, this region corresponds to an epitope or portionthereof of BA001 on CD137. Inspection of the sequences of human andcynomolgus monkey CD137, to both of which BA001 binds strongly (FIGS. 1Aand 1B), revealed complete sequence identity in the region describedabove (FIG. 11). By contrast, BA001 does not bind to any significantextent to murine CD137 (data not shown), which includes a number ofamino acid substitutions and insertions relative to human CD137 in thisregion (FIG. 14A). Lastly, a fragment of CD137, residues 26-63(DPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCD, SEQ ID NO: 34), also showeddeuterium protection. Without wishing to be bound by theory, it iscontemplated that this signal reflects CD137 dimerization via aPLAD-PLAD interaction, which may be enhanced by each arm ofBA001-F(ab′)₂ binding to, e.g., one of two distinct CD137 molecules,thereby bringing the PLAD domains of the CD137 molecules into closeenough proximity to permit PLAD-PLAD interaction.

Epitope Binding of Anti-Human CD137 Fab

Most of the CD137 peptides displayed identical or similar deuteriumlevels with and without BA001-Fab present. Several peptide segments,however, were found to have significantly decreased deuteriumincorporation upon BA001-Fab binding. All the residues in this paragraphare numbered according to SEQ ID NO: 25. The region defined by residues125-141 (FNDQKRGICRPWTNCSL, SEQ ID NO: 26) experienced strong deuteriumprotection when human CD137 was bound to BA001-Fab. This region thuscorresponds to an epitope or portion thereof of BA001 on CD137. Twoadditional regions, consisting of residues 89-98 (TPGFHCLGAG, SEQ ID NO:28), and residues 107-112 (KQGQEL, SEQ ID NO: 29), also exhibitedsubstantial deuterium protection, and thus optionally correspond toadditional epitopes, or portions thereof, of BA001 on CD137. Inspectionof the sequences of human and cynomolgus monkey CD137, to both of whichBA001 binds strongly (FIGS. 1A and 1B), revealed complete sequenceidentity in the regions corresponding to SEQ ID NO: 26 and 29, asdescribed above (FIG. 13). BA001 does not bind to any significant extentto murine CD137 (data not shown), which differs substantially from humanCD137 at these regions (FIG. 14A). Four amino acid substitutions werefound in the region of the cynomolgus monkey sequence corresponding toSEQ ID NO: 28 (i.e., T82I, P83S, F85Y, and G91E). The region of CD137consisting of residues 26-63 (SEQ ID NO: 34) did not exhibit anydeuterium protection in this experiment. Without wishing to be bound bytheory, it is contemplated that that binding of individual BA001-Fabfragments to single molecules of CD137 does not promote PLAD-PLADdimerization.

6.5.2 Epitope Mapping of Anti-CD137 Antibody Using Human/Mouse ChimericProteins

The epitope on human CD137 recognized by anti-CD137 antibody BA001 wasfurther studied using a series of murine switch mutant constructstransfected into Jurkat cells, which could then be analyzed by FACS.Jurkat switch mutants were generated that each constitutively expresseda human CD137 containing a single mutated region within theextracellular domain, in which that portion of the human CD137 sequencewas switched with the corresponding sequence from murine CD137 (i.e.,mutants 5014-5018 shown in FIG. 14A; sequences provided in Table 5below).

TABLE 5 Extracellular domain of human-mouse fusion constructsequences for CD137. SEQ ID Description Amino Acid Sequence* NO:Extracellular LQDPCSNCPAGTFCRKYNPVCKSCPPSTFSSAGGQRT 35 domain of human-CDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGC mouse fusionSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRP construct 5014WTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSV TPPAPARPGHSPQ ExtracellularLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSIGGQPNC 36 domain of human-NICRVCAGYFRFKKFCSSTSNAECDCTPGFHCLGAGC mouse fusionSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRP construct 5015WTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSV TPPAPAREPGHSPQ ExtracellularLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRT 37 domain of human-CDICRQCKGVFRTRKECSSTHNAECECIEGFHCLGPQ mouse fusionCTRCEKDCRPGQELTKKGCKDCCFGTFNDQKRGICR construct 5016PWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASS VTPPAPAREPGHSPQ ExtracellularLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRT 38 domain of human-CDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGC mouse fusionSMCEQDCKQGQELTKQGCKTCSLGTFNDQNGTGVC construct 5017RPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGAS SVTPPAPAREPGHSPQ ExtracellularLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRT 39 domain of human-CDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGC mouse fusionSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRP construct 5018WTNCSLDGRSVLKTGTTEKDVVCGPSPADLSPGASS VTPPAPAREPGHSPQ *Human CD137sequences are indicated in plain text. Murine CD137 sequences arebolded.

These engineered mutant cell lines were used to test whether anti-CD137antibody can bind to particular switch mutants. Absence of binding wouldthereby indicate possible epitope locations. The cell binding assayswere performed generally as described in Section 1.1. Briefly,transfected Jurkat cells were stained at 5×10⁴ cells/well using serialdilutions of anti-CD137 antibody (i.e., BA001, reference anti-CD137antibody #1, or reference anti-CD137 antibody #2) in a 96-well plate for25 minutes at 4° C. The cells were washed twice and incubated withF(ab′)₂ goat anti-human IgG-PE secondary antibody (JacksonImmunoResearch, Cat #109-116-098). The cells were then washed andsuspended in 80 μl of 2% paraformaldehyde (Electron Microscopy Sciences)prepared in PBS. Data were collected with BD FACS Canto and analyzedusing BD FACSDiva software.

As shown in FIG. 14B, antibody BA001 was able to bind to Jurkat cellsexpressing all of the murine switch mutants except mutant 5017, in whichthe sequence LTKKGCKDCCFGTFNDQKRGICRPWTNC (SEQ ID NO: 30) in human CD137was replaced with a corresponding region in murine CD137. The bindingpattern exhibited by BA001 is distinct from those exhibited by thereference anti-CD137 antibodies #1 and #2 (FIG. 14B). Referenceanti-CD137 antibody #1 exhibited minimal binding to mutant 5017 at lowerantibody concentrations, but binding was clearly detected atconcentrations at or greater than 10 μg/ml. In addition, differing fromBA001, reference anti-CD137 antibody #1 showed no binding to mutant5016.

The BA001 epitope in human CD137 identified from the murine switchmutants substantially overlapped with the BA001 epitope identified inthe HDX epitope mapping experiments described in Section 6.5.1. In theoverlapping region, four continuous amino acid residues of human CD137,having the sequence of KRGI (SEQ ID NO: 43), were different from thesequence of NGTGV (SEQ ID NO: 44) found in the corresponding region ofmurine CD137 (FIG. 15A). This difference could account for the lack ofsubstantial affinity of BA001 to murine CD137. To determine whether thesequence of KRGI (SEQ ID NO: 43) was an epitope recognized by BA001, twoproteins comprising chimeric CD137 extracellular domains were generated:the “4-aa human to mouse” CD137 protein was a human CD137 extracellulardomain having the KRGI sequence replaced with NGTGV; the “4-aa mouse tohuman” CD137 protein was a murine CD137 extracellular domain having theNGTGV sequence replaced with KRGI. These chimeric proteins furthercomprised a Gly-Ser linker and a 6×His tag at the C-terminus. Thesequences of the extracellular domains are provided in Table 6.

TABLE 6 Extracellular domain of chimeric CD137 proteins andfragments thereof SEQ ID Description Amino Acid Sequence* NO:Murine CD137 NGTGV 44 fragment ExtracellularLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRT 45 domain of “4-aaCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGC human to mouse”SMCEQDCKQGQELTKKGCKDCCFGTFNDQNGTGVCR CD137PWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASS VTPPAPAREPGHSPQ ExtracellularVQNSCDNCQPGTFCRKYNPVCKSCPPSTFSSIGGQPNC 46 domain of “4-aaNICRVCAGYFRFKKFCSSTHNAECECIEGFHCLGPQCT mouse to human”RCEKDCRPGQELTKQGCKTCSLGTFNDQKRGICRPWT CD137NCSLDGRSVLKTGTTEKDVVCGPPVVSFSPSTTISVTPE GGPGGHSLQVL

The chimeric CD137 proteins described above were tested in a surfaceplasmon resonance (SPR) assay. Briefly, a CM5 sensor chip was firstcoated with an anti-human Fab antibody using the amine coupling kit. 6μg/ml of BA001 and reference anti-CD137 antibody #1 was captured at aflow rate of 10 μl/min on flow-cells 2 and 3, respectively, keeping theflow-cell 1 as reference. A fully human CD137 protein, the “human tomouse” CD137 chimeric protein, and the “mouse to human” CD137 chimericprotein were then independently run at a concentration of 60 nM over theflow-cells at 30 μl/min for 90 seconds followed by a dissociation of 400seconds. Sensorgrams were recorded during both the association anddissociation phases.

As shown in FIG. 15B, when the KRGI (SEQ ID NO: 43) sequence of humanCD137 was replaced by the NGTGV (SEQ ID NO: 44) sequence of murineCD137, the chimeric protein lost its ability to bind BA001. Conversely,when the NGTGV (SEQ ID NO: 44) sequence of murine CD137 was replaced bythe KRGI (SEQ ID NO: 43) sequence of human CD137, the chimeric proteingained the ability to bind BA001. These data suggested that the KRGI(SEQ ID NO: 43) sequence represented the critical epitope region ofhuman CD137 involved in the binding to BA001.

In comparison, as shown in FIG. 15C, when the KRGI (SEQ ID NO: 43)sequence of human CD137 was replaced by the NGTGV (SEQ ID NO: 44)sequence of murine CD137, the chimeric protein lost its ability to bindreference anti-CD137 antibody #1. However, when the NGTGV (SEQ ID NO:44) sequence of murine CD137 was replaced by the KRGI (SEQ ID NO: 43)sequence of human CD137, the chimeric protein did not gain the abilityto bind reference anti-CD137 antibody #1. These data suggested that theKRGI (SEQ ID NO: 43) sequence, despite being necessary for the bindingof reference anti-CD137 antibody #1, was not sufficient in the murineCD137 context.

6.6 Characterization of Anti-CD137 Antibody Variants

This example describes the characterization of anti-CD137 antibodiesthat are variants of the BA001 antibody. The sequence information of thevariable regions of four of these antibodies is provided in Tables 1 and2.

6.6.1 BA001 Variants Bind to Human and Cynomolgus CD137.

Variants of BA001 were generated by screening scFv phage displaylibraries containing amino acid substitutions in CDRH1, CDRH3, and CDRL3of BA001. Briefly, an scFv of BA001 comprising the amino acid sequenceof SEQ ID NO: 55 was generated, mutagenesis of the scFv was conducted,and the positive clones were enriched by affinity-based selection. Atotal of 347 clones that bound to human CD137 were identified. ConsensusCDRH1, CDRH3, and CDRL3 sequences constructed from analysis of the aminoacid sequence of the 347 clones are set forth in SEQ ID NOs: 82, 83, and84, respectively. Among the 347 clones, 233 had a dissociation rate ofless than 1×10⁻³ s⁻¹. Consensus CDRH1, CDRH3, and CDRL3 sequencesconstructed from these 233 clones are set forth in SEQ ID NOs: 85, 86,and 87, respectively.

The binding affinities of four of the BA001 variants to CD137 werefurther characterized. These four variants, named BA049, BA050, BA051,and BA052, comprised the scFv amino acid sequences set forth in SEQ IDNOs: 69, 70, 71, and 72, respectively, as provided in Table 1. The fourvariants were converted to an IgG1 format, and their heavy chain andlight chain sequences are provided in Tables 1 and 2. To measure bindingaffinities, extracellular domains of human CD137, cynomolgus CD137,mouse-human fusion construct 5015 (murine CD137 with amino acid residues53-81 replaced with the corresponding sequence of human CD137), andmouse-human fusion construct 5017 (murine CD137 with amino acid residues112-140 replaced with the corresponding sequence of human CD137), eachcomprising a Gly-Ser linker followed by a 6×His tag, were used asantigens. The extracellular domain sequences of the two chimericproteins (excluding the linker and 6×His tag) are provided in Table 7.

TABLE 7 Extracellular domain of chimeric CD137 proteins. SEQ IDDescription Amino Acid Sequence* NO: ExtracellularVQNSCDNCQPGTFCRKYNPVCKSCPPSTFSSAGGQRT 47 domain of mouse-CDICRQCKGVFRTRKECSSTHNAECECIEGFHCLGPQ human fusionCTRCEKDCRPGQELTKQGCKTCSLGTFNDQNGTGVCR construct 5015PWTNCSLDGRSVLKTGTTEKDVVCGPPVVSFSPSTTIS VTPEGGPGGHSLQVL ExtracellularVQNSCDNCQPGTFCRKYNPVCKSCPPSTFSSIGGQPNC 48 domain of mouse-NICRVCAGYFRFKKFCSSTHNAECECIEGFHCLGPQCT human fusionRCEKDCRPGQELTKKGCKDCCFGTFNDQKRGICRP construct 5017WTNCSLDGRSVLKTGTTEKDVVCGPPVVSFSPSTTISV TPEGGPGGHSLQVL

The affinities of the BA001 variants (in the IgG1 format) to theantigens were measured by ELISA. Specifically, 50 μl of 5 μg/ml of eachantigen diluted in 1×PBS pH 7.4 (Gibco™, Cat no. 10010056) was added toeach well in a Thermo Scientific™ Black 96-Well Immuno Plate(Thermofisher Scientific, Cat no. 437111) and incubated at 4° C.overnight. The plate was washed three times with PBS using the Biotek405TS microplate washer with Biostack3 microplate stacker. The plate wasblocked by incubating with 300 ul/well of 3% Milk Powder in PBS (Marveldried skimmed milk powder) for 1 hour at room temperature, and werewashed three times with 1×PBS. The antibodies titrated in 3% M-PBS (MilkPowder in 1x PBS) were added to the plate and incubated for 1 hour atroom temperature. The plate was washed three times with 1×PBS with 0.1%Tween20 (Sigma Aldrich, Cat No. P1379) and three times with 1×PBS usingthe plate washer. 50 μl Biotin-SP (long spacer) AffiniPure GoatAnti-Human IgG, Fcγ Fragment Specific (Jackson Immuno Research,Code:109-065-098, Lot no. 123909) diluted at 1:2000 in 3% M-PBS wasadded to each well and incubated for 1 hour at room temperature. Theplate was washed three times with 1×PBS with 0.1% Tween20 and threetimes with 1×PBS using the plate washer. For detection, 50 μl of DELFIA®Europium-labeled streptavidin (PerkinElmer, Part no. 1244-360, Lot no.2195997) diluted in the DELFIA® Assay buffer (PerkinElmer, Part no.4002-0010, Lot no. 646702) at 1:500 was added to each well and incubatedfor 1 hour at room temperature. The plate was washed three times with1×PBS with 0.1% Tween20 and three times with PBS using the plate washer.50 ul of DELFIA® enhancement solution (PerkinElmer, Part no. 4001-0010,Lot no. 650872) was added to each well, and incubated at roomtemperature for 5 mins with gentle shaking. Fluorescence was read atExcitation 340 nm and Emission 615 nm using Tecan Infinite M1000 Proplate reader. The data was acquired with the Tecan iControl softwareversion 1.11.1.0, and analyzed with Graphpad Prism version 7.02.

As shown in FIGS. 16A and 16B, the four BA001 variants showed binding tohuman and cynomolgus CD137. Additionally, they bound to mouse-humanfusion construct 5017 (“mCD137-human112-139”) (FIG. 16C) but notmouse-human fusion construct 5015 (“mCD137-human53-80”) (FIG. 16D),indicating that they bound to an epitope of human CD137 in the region ofamino acid residues 112-139. These data suggest that these four variantsbound to the same or a similar epitope as BA001.

The invention is not to be limited in scope by the specific embodimentsdescribed herein. Indeed, various modifications of the invention inaddition to those described will become apparent to those skilled in theart from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references (e.g., publications or patents or patent applications)cited herein are incorporated herein by reference in their entiretiesand for all purposes to the same extent as if each individual reference(e.g., publication or patent or patent application) was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

Other embodiments are within the following claims.

1.-86. (canceled)
 87. An isolated antibody that specifically binds tohuman CD137, the antibody comprising a VH comprising the CDRH1, CDRH2,and CDRH3 amino acid sequences of the VH amino acid sequence of SEQ IDNO: 7, and a VL comprising the CDRL1, CDRL2, and CDRL3 amino acidsequences of the VL amino acid sequence of SEQ ID NO: 8, wherein theantibody is conjugated to a cytotoxic agent, cytostatic agent, toxin,radionuclide, detectable label, or a second antibody. 88.-93. (canceled)94. A method of increasing an immune response in a subject, the methodcomprising administering to the subject an effective amount of anantibody that specifically binds to human CD137, the antibody comprisinga VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of theVH amino acid sequence of SEQ ID NO: 7, and a VL comprising the CDRL1,CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence ofSEQ ID NO: 8, or a pharmaceutical composition thereof.
 95. A method oftreating cancer in a subject, the method comprising administering to thesubject an effective amount of an antibody that specifically binds tohuman CD137, the antibody comprising a VH comprising the CDRH1, CDRH2,and CDRH3 amino acid sequences of the VH amino acid sequence of SEQ IDNO: 7, and a VL comprising the CDRL1, CDRL2, and CDRL3 amino acidsequences of the VL amino acid sequence of SEQ ID NO: 8, or apharmaceutical composition thereof.
 96. The method of claim 94, whereinthe antibody or pharmaceutical composition thereof is administeredsystemically.
 97. The method of claim 96, wherein the antibody orpharmaceutical composition thereof is administered intravenously. 98.The method of claim 94, wherein the antibody or pharmaceuticalcomposition thereof is administered subcutaneously, intratumorally, oris delivered to a tumor draining lymph node.
 99. The method of claim 94,further comprising administering an additional therapeutic agent to thesubject.
 100. The method of claim 99, wherein the additional therapeuticagent is a chemotherapeutic agent, a checkpoint targeting agent, aninhibitor of indoleamine-2,3-dioxygenase (IDO), or a vaccine. 101.(canceled)
 102. The method of claim 100, wherein: (a) the checkpointtargeting agent is selected from the group consisting of an antagonistanti-PD-1 antibody, an antagonist anti-PD-L1 antibody, an antagonistanti-PD-L2 antibody, an antagonist anti-CTLA-4 antibody, an antagonistanti-TIM-3 antibody, an antagonist anti-LAG-3 antibody, an antagonistanti-VISTA antibody, an antagonist anti-CD96 antibody, an antagonistanti-CEACAM1 antibody, an antagonist anti-TIGIT antibody, an agonistanti-GITR antibody, and an agonist anti-OX40 antibody; (b) the inhibitorof IDO is selected from the group consisting of epacadostat, F001287,indoximod, and NLG919; (c) the vaccine comprises a heat shock proteinpeptide complex (HSPPC) comprising a heat shock protein complexed withan antigenic peptide, optionally wherein the heat shock protein is hsc70or gp96 protein and the antigenic peptide is a tumor-associatedantigenic peptide. 103.-109. (canceled)
 110. A method of treating aninfectious disease in a subject, the method comprising administering tothe subject an effective amount of an antibody that specifically bindsto human CD137, the antibody comprising a VH comprising the CDRH1,CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence ofSEQ ID NO: 7, and a VL comprising the CDRL1, CDRL2, and CDRL3 amino acidsequences of the VL amino acid sequence of SEQ ID NO: 8, or apharmaceutical composition thereof.
 111. The method of claim 94, whereinthe antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively.112. The method of claim 94, wherein the VH comprises the amino acidsequence of SEQ ID NO: 7, and/or the VL comprises the amino acidsequence of SEQ ID NO:
 8. 113. The method of claim 94, wherein theantibody comprises a heavy chain constant region selected from the groupconsisting of human IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂.
 114. Themethod of claim 113, wherein the heavy chain constant region is an IgG₁heavy chain constant region.
 115. The method of claim 113, wherein: (a)the amino acid sequence of the IgG₁ constant region comprises an N297Amutation; (b) the amino acid sequence of the IgG₁ constant regioncomprises S267E and L328F mutations; (c) the amino acid sequence of theIgG₂ constant region comprises a N297A mutation; or (d) the amino acidsequence of the IgG₄ constant region comprises an S228P mutation. 116.The method of claim 113, wherein the heavy chain constant regioncomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 15-20.
 117. The method of claim 94, wherein the antibodycomprises a light chain constant region selected from the groupconsisting of a human kappa light chain constant region and a humanlambda light chain constant region.
 118. The method of claim 117,wherein the light chain constant region comprises the amino acidsequence of SEQ ID NO:
 22. 119. The method of claim 94, wherein theantibody comprises a heavy chain comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 9-14 and 49-54, and/ora light chain comprising the amino acid sequence of SEQ ID NO:
 21. 120.The method of claim 119, wherein the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 9, and a light chaincomprising the amino acid sequence of SEQ ID NO: 21.